Disulfide bridge-dependent dimerization triggers FGF2 membrane translocation into the extracellular space
- Heidelberg University Biochemistry Center, Germany
- Department of Physics, University of Helsinki, Finland
- Schaller Research Group, Department of Infectious Diseases-Virology, Heidelberg University Hospital, Germany
- J. Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences, Czech Republic
- Institute for Chemistry and Biochemistry, Freie Universität Berlin, Germany
Figures
Figure 1
Cysteine residues in positions 77 and 95 of fibroblast growth factor 2 (FGF2) play a role in its unconventional secretion from cells.
(A) Representative wide-field and TIRF images of real-time single molecule TIRF recruitment assay conducted on stable CHO K1 cell lines overexpressing either wild-type (WT) or mutant (C77A, C95A, C77/95A) FGF2-GFP in a doxycycline-dependent manner. Beyond cell lines expressing various forms of FGF2-GFP, a GFP-expressing cell line was used to subtract GFP background. Wide-field images show the overall FGF2-GFP (or GFP) expression levels. Single FGF2-GFP (or GFP) particles recruited at the inner plasma membrane leaflet and detected within the TIRF field are highlighted with a pink circle. (B) Quantification of real-time single molecule TIRF recruitment assay conducted on the cell lines shown in panel A. Recruitment efficiency at the inner plasma membrane leaflet of FGF2-GFP WT was set to 1. Each square represents a single cell. Mean recruitment efficiency values are shown in brackets. Data are shown as mean with standard deviations (n=4). Statistical analysis was based on a one-way ANOVA test performed in Prism (version 9.4.1), ***p≤0.001. (C) Representative western blot of cell surface biotinylation assay conducted on stable CHO cell lines overexpressing either WT or mutant (C77A, C95A, C77/95A, C77S, C95S, C77/95S) FGF2-GFP in a doxycycline-dependent manner. Total cellular proteins and biotinylated surface proteins were analyzed. The analysis was conducted against GFP, to detect the various FGF2-GFP mutant forms, and GAPDH, both as a loading and a cellular integrity control. (D) Quantification of cell surface biotinylation assay conducted on the cell lines shown in panel D. Secretion efficiency of FGF2-GFP WT was set to 100%. Mean secretion efficiency values for each cell line are shown in brackets. Data are shown as mean with standard deviations (n=4). Statistical analysis was based on a one-way ANOVA test performed in Prism (version 9.4.1), not significant (ns) p>0.05, ***p≤0.001.
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Figure 1—source data 1
Real-time single molecule TIRF recruitment assay.
- https://cdn.elifesciences.org/articles/88579/elife-88579-fig1-data1-v1.zip
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Figure 1—source data 2
Original file for the western blot analysis in Figure 1C (cell surface biotinylation assay).
- https://cdn.elifesciences.org/articles/88579/elife-88579-fig1-data2-v1.zip
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Figure 1—source data 3
PDF containing Figure 1C and original scans of the relevant western blot analysis.
- https://cdn.elifesciences.org/articles/88579/elife-88579-fig1-data3-v1.zip
Figure 2
Formation of higher fibroblast growth factor 2 (FGF2) oligomers on the membrane surface of giant unilamellar vesicles (GUVs) depends on C95.
(A) Oligomeric size distribution of FGF2-GFP variants. GUVs with a plasma membrane-like lipid composition containing 2 mol% phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) were incubated with variant forms of His-tagged FGF2-Y81pCMF-GFP as indicated. The oligomer size was determined by brightness analysis as described in detail in Materials and methods. Each dot corresponds to a data point measured on a single GUV with the number of GUVs analyzed n (wild-type [WT]; n=68, C77A; n=42, C95A; n=31, C77/95A; n=9). Mean values with standard deviations are shown. One-way ANOVA with Tukey’s post hoc test was performed in Prism (version 9.4.1). Mean values are shown in brackets, not significant (ns) p>0.05, ****p≤0.0001. Data distribution was assumed to be normal, but this was not formally tested. (B) Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of FGF2-Y81pCMF-GFP variant forms indicated. Purified proteins were analyzed for homogeneity using Coomassie staining.
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Figure 2—source data 1
Oligomeric size distribution of fibroblast growth factor 2 (FGF2)-GFP variants.
- https://cdn.elifesciences.org/articles/88579/elife-88579-fig2-data1-v1.zip
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Figure 2—source data 2
Original file for the blot analysis in Figure 2B.
- https://cdn.elifesciences.org/articles/88579/elife-88579-fig2-data2-v1.zip
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Figure 2—source data 3
PDF containing Figure 2B and original scans of the relevant blot analysis.
- https://cdn.elifesciences.org/articles/88579/elife-88579-fig2-data3-v1.zip
Figure 3
Fibroblast growth factor 2 (FGF2) dimer formation in cells depends on C95 as revealed by chemical cross-linking.
Using cellular lysates, FGF2 dimer formation was analyzed by chemical cross-linking. The FGF2 variants (wild-type [WT], C77A, C95A, and C77/95A) were transiently expressed in HeLa S3 cells as constructs connecting the FGF2 open reading frame with GFP via a P2A site, producing stoichiometric amounts of untagged FGF2 and GFP, the latter used to label transfected cells. The corresponding cellular lysates were treated with three different cross-linkers: PMPI (N-p-maleimidophenylisocyanate; bifunctional cross-linker with a spacer length of 8.7 Å targeting sulfhydryl groups at one end [maleimide] and hydroxyl groups at the other end [isocyanate], A and D), BMOE ([bismaleimidoethane; bifunctional maleimide-based cross-linker with a short 8 Å spacer length targeting sulfhydryl groups], B and E), or BMH ([bismaleimidohexane; bifunctional maleimide-based cross-linker with a long 13 Å spacer length targeting sulfhydryl groups], C and F), respectively. Cross-linking products were analyzed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by western blotting using polyclonal anti-FGF2 antibodies. (A, B, C) Representative examples of the western analyses for each of the three cross-linkers described above. FGF2 monomers (18 kDa) are labeled with ‘♦’, FGF2 dimers (36 kDa) with ‘♦♦’ and small amounts of monomeric full-length FGF2-P2A-GFP (∼50 kDa) with ‘◊’. (D, E, F) Quantification of FGF2 dimer to FGF2 monomer ratios. Signal intensities were quantified using a LI-COR Odyssey CLx imaging system. The FGF2 dimer to monomer ratios were determined in four independent experiments with the standard error of the mean shown, not significant (ns) p>0.05, *p≤0.05, **p≤0.01, ***p≤0.001. Statistical analyses were based on a two-tailed, unpaired t-test using GraphPad Prism (version 9.4). Data distribution was assumed to be normal, but this was not formally tested.
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Figure 3—source data 1
Cross-linking quantification of fibroblast growth factor 2 (FGF2) dimer to FGF2 monomer ratios.
- https://cdn.elifesciences.org/articles/88579/elife-88579-fig3-data1-v1.zip
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Figure 3—source data 2
Original file for the western blot analysis in Figure 3A.
- https://cdn.elifesciences.org/articles/88579/elife-88579-fig3-data2-v1.zip
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Figure 3—source data 3
PDF containing Figure 3A and original scans of the relevant western blot analysis.
- https://cdn.elifesciences.org/articles/88579/elife-88579-fig3-data3-v1.zip
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Figure 3—source data 4
Original file for the western blot analysis in Figure 3B.
- https://cdn.elifesciences.org/articles/88579/elife-88579-fig3-data4-v1.zip
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Figure 3—source data 5
PDF containing Figure 3B and original scans of the relevant western blot analysis.
- https://cdn.elifesciences.org/articles/88579/elife-88579-fig3-data5-v1.zip
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Figure 3—source data 6
Original file for the western blot analysis in Figure 3C.
- https://cdn.elifesciences.org/articles/88579/elife-88579-fig3-data6-v1.zip
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Figure 3—source data 7
PDF containing Figure 3C and original scans of the relevant western blot analysis.
- https://cdn.elifesciences.org/articles/88579/elife-88579-fig3-data7-v1.zip
Figure 4
Membrane pore formation triggered by fibroblast growth factor 2 (FGF2) oligomers depends on C95.
Carboxyfluorescein was sequestered in large unilamellar liposomes containing a plasma membrane-like lipid composition including 2 mol% phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). (A) Liposomes were incubated with the His-tagged FGF2-Y81pCMF (2 µM)-based variant forms indicated (wild-type [WT], C77A, C95A, and C77/95A). Membrane pore formation was analyzed by measuring the release of luminal carboxyfluorescein quantified by fluorescence dequenching as detailed in Materials and methods. The results shown are representative for three independent experiments. (B) Quality of the various recombinant proteins was analyzed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining.
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Figure 4—source data 1
Membrane pore formation triggered by fibroblast growth factor 2 (FGF2) oligomers.
- https://cdn.elifesciences.org/articles/88579/elife-88579-fig4-data1-v1.zip
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Figure 4—source data 2
Original file for the blot analysis in Figure 4B.
- https://cdn.elifesciences.org/articles/88579/elife-88579-fig4-data2-v1.zip
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Figure 4—source data 3
PDF containing Figure 4B and original scans of the relevant blot analysis.
- https://cdn.elifesciences.org/articles/88579/elife-88579-fig4-data3-v1.zip
Figure 5
Full membrane translocation of fibroblast growth factor 2 (FGF2) across giant unilamellar vesicle (GUV) lipid bilayers depends on C95.
Reconstitution of FGF2 membrane translocation with purified components. GUVs with a plasma membrane-like lipid composition containing 2 mol% phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) were prepared in the presence or absence of long-chain heparins as described in detail in Materials and methods. In brief, Rhodamine-PE (Rhod.-PE) was incorporated into the lipid bilayer during GUV preparation as membrane marker. After removal of excess heparin by low-speed centrifugation, GUVs were incubated with His-tagged FGF2-Y81pCMF-GFP (200 nM) variants as indicated and a small fluorescent tracer (Alexa647). Following 180 min of incubation luminal penetration of GUVs by FGF2-Y81pCMF-GFP and small tracer molecules was analyzed by confocal microscopy. (A) Radial intensity profiles of representative examples quantifying GFP fluorescence in the GUV lumen versus the exterior for FGF2-Y81pCMF-GFP wild-type (WT) (subpanel a), C77A (subpanel b), C95A (subpanel c), and C77/95A (subpanel d). (B) Quantification and statistical analysis of FGF2 membrane translocation and membrane pore formation. Gray bars indicate the percentage of GUVs with membrane pores with a ratio of Alexa647 tracer fluorescence in the lumen versus the exterior of ≥0.6. Green bars indicate the percentage of GUVs where membrane translocation of GFP-tagged proteins had occurred with a ratio of GFP fluorescence in the lumen versus the exterior of ≥1.6 being used as a threshold value. Each dot represents an independent experiment each of which involved the analysis of 20–120 GUVs per experimental condition. Mean values with standard deviations are shown. Statistical analyses are based on two-tailed, unpaired t-test performed in Prism (version 9.4.1), not significant (ns) p>0.05, *p≤0.05. Data distribution was assumed to be normal, but this was not formally tested. For details, see Materials and methods. (C) Representative confocal images of plasma membrane-like GUVs containing PI(4,5)P2 and long-chain heparins in the lumen after 180 min incubation with His-tagged FGF2-Y81pCMF-GFP (200 nM) variants as indicated and a small fluorescent tracer (Alexa647; scale bar = 10 µm).
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Figure 5—source data 1
Fibroblast growth factor 2 (FGF2) membrane translocation and membrane pore formation assay (phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]) containing liposomes.
- https://cdn.elifesciences.org/articles/88579/elife-88579-fig5-data1-v1.zip
Figure 6
Fibroblast growth factor 2 (FGF2) membrane translocation across giant unilamellar vesicle (GUV) lipid bilayers is abrogated when phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) is substituted by a Ni-NTA lipid used to recruit His-tagged FGF2 fusion proteins.
GUVs with a plasma membrane-like lipid composition containing 2 mol% Ni-NTA-lipid anchor were prepared in the presence or absence of long-chain heparins. Luminal penetration of GUVs by FGF2-Y81pCMF-GFP was analyzed by confocal microscopy as described in the legend to Figure 5. (A) Radial intensity profiles of representative examples quantifying GFP fluorescence in the GUV lumen versus the exterior for FGF2-Y81pCMF-GFP wild-type (WT) (subpanel a), C77A (subpanel b), C95A (subpanel c), and C77/95A (subpanel d). (B) Quantification and statistical analysis of FGF2 membrane translocation and membrane pore formation. Gray bars indicate the percentage of GUVs with membrane pores with a ratio of Alexa647 tracer fluorescence in the lumen versus the exterior of ≥0.6. Green bars indicate the percentage of GUVs where membrane translocation of GFP-tagged proteins had occurred with a ratio of GFP fluorescence in the lumen versus the exterior of ≥1.6 being used as a threshold value. Each dot represents an independent experiment each of which involved the analysis of 20–120 GUVs per experimental condition. Mean values with standard deviations are shown. Statistical analyses are based on two-tailed, unpaired t-test performed in Prism (version 9.4.1), not significant (ns) p>0.05. Data distribution was assumed to be normal, but this was not formally tested. (C) Representative confocal images of plasma membrane-like GUVs containing Ni-NTA-lipid anchor and long-chain heparins in the lumen after 180 min incubation with His-tagged FGF2-Y81pCMF-GFP (200 nM) variants as indicated and a small fluorescent tracer (Alexa647; scale bar = 10 µm).
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Figure 6—source data 1
Fibroblast growth factor 2 (FGF2) membrane translocation and membrane pore formation assay (Ni-NTA containing liposomes).
- https://cdn.elifesciences.org/articles/88579/elife-88579-fig6-data1-v1.zip
Figure 7
C77 is a component of the protein-protein interaction surface between fibroblast growth factor 2 (FGF2) and the α1 subunit of the Na,K-ATPase.
Kinetic analysis of the direct interaction of FGF2 with α1-subCD3 (Legrand et al., 2020). (A) FGF2 binds to α1 with K54, K60, and C77 being part of the protein-protein interaction interface (figure adapted from Legrand et al., 2020). (B) FGF2 directly binds to α1 in a dose-dependent manner. Biolayer interferometry (BLI) allows temporal resolution of association and dissociation. Biotinylated Hisα1-subCD3-WT protein was immobilized on Streptavidin sensors followed by incubation with His-tagged FGF2 wild-type protein (HisFGF2-WT) at concentrations indicated. The data shown is representative of three independent experiments. Data were analyzed with Data Analysis HT 12.0 software (Sartorius) using a 1:1 binding model. See Materials and methods for details. Mean with standard deviations of KD, ka, and kd values (n=3) are given. (C, D, E) Comparison of FGF2 variants. (C) BLI measurements were conducted using immobilized Hisα1-subCD3 with FGF2 variants (1000 nM concentration) as indicated. The data shown is representative for four independent experiments. (D) The quality of HisFGF2 proteins was analyzed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining. 3 µg of each variant were loaded as indicated. (E) Phase shift at time point 600 s. Mean values with standard deviations of four independent experiments are shown. One-way ANOVA with Tukey’s post hoc test was performed in Prism (version 9.4.1). Mean values are shown in brackets, not significant (ns) p>0.5, ****p≤0.0001. Data distribution was assumed to be normal, but this was not formally tested.
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Figure 7—source data 1
Kinetic analysis of fibroblast growth factor 2 (FGF2)/α1-subCD3 interaction.
- https://cdn.elifesciences.org/articles/88579/elife-88579-fig7-data1-v1.zip
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Figure 7—source data 2
Original file for the blot analysis in Figure 7D.
- https://cdn.elifesciences.org/articles/88579/elife-88579-fig7-data2-v1.zip
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Figure 7—source data 3
PDF containing Figure 7D and original scans of the relevant blot analysis.
- https://cdn.elifesciences.org/articles/88579/elife-88579-fig7-data3-v1.zip
Figure 8
Simulations reveal that the C95-C95 interaction interface forms independently of the disulfide bridge.
(A) The distribution of the C95-C95 and C77-C77 distance shows that only the former can come within 1 nm of each other during the unbiased 360 molecular dynamics (MD) simulations, where one fibroblast growth factor 2 (FGF2) monomer was systematically rotated to explore all possible C95-involved dimerization interfaces. (B) The C-alpha atoms of the two monomers were reduced to a two-dimensional (2D) representation using an orthogonal autoencoder. The points are colored with the C95-C95 distance. A cluster with low C95-C95 distance is visible in the representation. (C) The encoded space was clustered with a Bayesian Gaussian mixture model (GMM) to find regions of distinct conformational structures. The eight identified clusters are indicated in the figure, along with the cluster mean shown in black dots and the corresponding cluster label. (D) Populations of the individual clusters in the GMM. Cluster 2 have the largest population among the eight identified clusters. (E) The C95-C95 distance indicates that the highest occupied cluster (Cluster 2) also has the highest likelihood of low C95-C95 distance (below 1 nm). (F) Representative structure of the FGF2 dimer from Cluster 2 showing C95 residues in proximity and crucial residues responsible for salt bridge interactions.
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Figure 8—source data 1
Populations of the individual clusters in the Bayesian Gaussian mixture model.
- https://cdn.elifesciences.org/articles/88579/elife-88579-fig8-data1-v1.zip
Figure 9
Characterization of C95 disulfide-bridged fibroblast growth factor 2 (FGF2) via molecular dynamics (MD) simulations.
(A) The AlphaFold2 model dimeric interface’s stability (subpanel a) was tested by conducting 1-µs-long MD simulations in water, which revealed the interface’s high flexibility (subpanel b). (B) Unbiased all-atom MD simulations were used to sample the FGF2 dimer-membrane interaction pathway, mediated via the experimentally known phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) binding pocket (K127, R128, K133). The free energy profile of FGF2 dimer-membrane interaction was determined from biased (umbrella sampling) MD simulations and plotted against the center of the mass distance of FGF2 dimer from phosphate atoms of the interacting membrane surface. Subpanels a–c show the interaction pathway’s initial, intermediate, and final states, while subpanel d shows the free energy profile. The statistical error was determined with 200 bootstrap analyses.
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Figure 9—source data 1
Umbrella sampling free energy profile.
- https://cdn.elifesciences.org/articles/88579/elife-88579-fig9-data1-v1.zip
Figure 10
Cross-linking mass spectrometry visualization of fibroblast growth factor 2 (FGF2) dimer interfaces.
(A) Overview of the inter-cross-linked fragments for His-tagged FGF2-WT and FGF2-C77/95A in the presence and absence of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-containing liposomes. (B) Liposome-induced cross-linking findings align seamlessly with molecular dynamics simulations’ C95-dependent dimer model interface. Fragments K74-K74, K85-K85, and K94-K94 can be positioned within the simulation model at a Cα-Cα distance below 23 Å. This concurs with the theoretical maximum Cα-Cα distance of approximately 26.4 Å for disuccinimidyldibutyric urea (DSBU)-linked lysine residues. (C) Cross-linking mass spectrometry data are compatible with an additional dimerization interface. It is incompatible with disulfide bridge formation but consistent with a membrane-bound FGF2 dimer configuration. Fragments K34-K34, K54-K54, K60-K60, and K143-K143 can be positioned at a Cα-Cα distance below 23 Å.
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Figure 10—source data 1
Cross-linking mass spectrometry fragments.
- https://cdn.elifesciences.org/articles/88579/elife-88579-fig10-data1-v1.zip
Figure 11 with 1 supplement
Cryo-electron tomography visualization of fibroblast growth factor 2 (FGF2) dimer in proteoliposomes.
(A) Example slices of cryo-electron tomograms (subpanels a–c; 2.3 nm thickness) showing His-FGF2-Y81pCMF-Halo bound to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-containing liposomes acquired at nominal defocus –3 µm. Magnified views from FGF2-Halo monomers, dimers, and higher oligomers (subpanels e–f). (B) Subtomogram average of the V-shaped FGF2-Halo dimer interacting with the membrane of PI(4,5)P2-containing liposomes (subpanels a–b). Subtomogram average of ‘V-shaped’ FGF2 dimers were manually picked using a dipole model in Dynamo (number of particles = 186). Top and side views are shown in subpanels a and b, respectively. (c–d) Three-dimensional (3D) map with manually fitted crystal structures of two Halo domains (PDB:4KAJ) and two FGF2 (PDB:1BFF) monomers. (e–f) Atom-scale molecular dynamics simulation model of V-shaped C95 disulfide-bridged FGF2-Halo dimer stable over 500 ns.
Figure 11—video 1
Simulation of the modeled V-shaped dimer interacting with the membrane surface over 500 ns.
Figure 12
Cryo-electron tomography visualization of fibroblast growth factor 2 (FGF2) dimer in proteoliposomes.
From a top and side view perspective, a comparison between the AlphaFold2 Multimer v3 model (panels A–B) and the one used for molecular dynamics (MD) simulations (panels C–D). The AlphaFold model accurately predicted the orientation of FGF2 dimer, with the phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) binding residues correctly positioned for a membrane-bound state (blue residues). However, the two cysteines 95 (red residues), although located at the interface, were observed to be distant. To improve the model, we replaced the FGF2 dimer with the C95-C95 disulfide-bridged dimer interface characterized with MD simulations (Figure 9). Furthermore, we used the ‘Fit in Map’ command provided by the ChimeraX software to locally optimize the fit of one of the two Halo domains' atomic coordinates into the density map.
Author response image 1
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | FGF2, fibroblast growth factor 2 | GenBank | Gene ID: 2247 | NP_001348594.1 18 kDa isoform |
| Gene (Homo sapiens) | ATP1A1, ATPase Na+/K+ transporting subunit alpha 1 | GenBank | Gene ID: 476 | |
| Strain, strain background (Escherichia coli) | W3110Z1 | Lutz and Bujard, 1997 | Chemically competent | |
| Strain, strain background (Escherichia coli) | BL21 Star (DE3) | Thermo Fisher | Chemically competent | |
| Cell line (Chinese Hamster) | CHO K1 FGF2-GFP (WT) | Legrand et al., 2020 | Parental Cell line CHO K1 MCAT Tam 2: Zehe et al., 2006 | |
| Cell line (Chinese Hamster) | CHO K1 FGF2-GFP (C77A) | This work | Parental Cell line CHO K1 MCAT Tam 2: Zehe et al., 2006 | |
| Cell line (Chinese Hamster) | CHO K1 FGF2-GFP (C95A) | This work | Parental Cell line CHO K1 MCAT Tam 2: Zehe et al., 2006 | |
| Cell line (Chinese Hamster) | CHO K1 FGF2-GFP (C77/95A) | Legrand et al., 2020 | Parental Cell line CHO K1 MCAT Tam 2: Zehe et al., 2006 | |
| Cell line (Chinese Hamster) | CHO K1 GFP | Legrand et al., 2020 | Parental Cell line CHO K1 MCAT Tam 2: Zehe et al., 2006 | |
| Cell line (Chinese Hamster) | CHO FGF2-GFP (WT) | Müller et al., 2015 | Parental Cell line CHO MCAT Tam 2: Engling et al., 2002 | |
| Cell line (Chinese Hamster) | CHO FGF2-GFP (C77A) | Müller et al., 2015 | Parental Cell line CHO MCAT Tam 2: Engling et al., 2002 | |
| Cell line (Chinese Hamster) | CHO FGF2-GFP (C95A) | Müller et al., 2015 | Parental Cell line CHO MCAT Tam 2: Engling et al., 2002 | |
| Cell line (Chinese Hamster) | CHO FGF2-GFP (C77/95A) | Müller et al., 2015 | Parental Cell line CHO MCAT Tam 2: Engling et al., 2002 | |
| Cell line (Chinese Hamster) | CHO FGF2-GFP (C77S) | This work | Parental Cell line CHO MCAT Tam 2: Engling et al., 2002 | |
| Cell line (Chinese Hamster) | CHO FGF2-GFP (C95S) | This work | Parental Cell line CHO MCAT Tam 2: Engling et al., 2002 | |
| Cell line (Chinese Hamster) | CHO FGF2-GFP (C77/95S) | This work | Parental Cell line CHO MCAT Tam 2: Engling et al., 2002 | |
| Cell line (Homo sapiens) | Hela S3 FGF2-P2A-GFP (WT) | This work | Parental Cell line Hela S3: Sparn et al., 2022b | |
| Cell line (Homo sapiens) | Hela S3 FGF2-P2A-GFP (C77A) | This work | Parental Cell line Hela S3: Sparn et al., 2022b | |
| Cell line (Homo sapiens) | Hela S3 FGF2-P2A-GFP (C95A) | This work | Parental Cell line Hela S3: Sparn et al., 2022b | |
| Cell line (Homo sapiens) | Hela S3 FGF2-P2A-GFP (C77/C95A) | This work | Parental Cell line Hela S3: Sparn et al., 2022b | |
| Antibody | Anti-GFP (rabbit polyclonal) | Custom-made, Pineda Antibody Service | Dilution (1:500) | |
| Antibody | Anti-GAPDH (mouse monoclonal) | Thermo Fisher Scientific | AM4300 | Dilution (1:20,000) |
| Antibody | Anti-rabbit – Secondary Antibody conjugated to IRDye 800CW (goat polyclonal) | LI-COR Biosciences | 926-32211 | Dilution (1:10,000) |
| Antibody | Anti-mouse – Secondary Antibody conjugated to Alexa Fluor 680 (goat polyclonal) | Thermo Fisher Scientific | A21057 | Dilution (1:10,000) |
| Recombinant DNA reagent | pEVOL-pCMF | Young et al., 2010 | ||
| Recombinant DNA reagent | pET15b-Hisα1-subCD3-WT | Legrand et al., 2020 | ||
| Recombinant DNA reagent | pET15b-HisFGF2-Y81pCMF-GFP | Steringer et al., 2017 | ||
| Recombinant DNA reagent | pQE30-HisFGF2 | Steringer et al., 2012 | ||
| Recombinant DNA reagent | pQE30-HisFGF2-Y81pCMF | Müller et al., 2015 | ||
| Commercial assay or kit | EZ-Link NHS-PEG4-Biotin | Thermo Scientific | A39259 | |
| Commercial assay or kit | Zeba Spin Desalting Columns | Thermo Scientific | 89882 | |
| Commercial assay or kit | Streptavidin sensors | Sartorius | SA biosensors, 18-5019 | |
| Chemical compound, drug | p-Carboxylmethylphenylalanine (pCMF) | ENAMINE Ltd, Kiev, Ukraine | ||
| Chemical compound, drug | Atto-633 labeled dioleoyl-PE [Atto-633-DOPE] | ATTO-TEC | ||
| Software, algorithm | GraphPad Prism, version 9.4 | GraphPad Prism | ||
| Software, algorithm | GROMACS, version 2022 | GROMACS | ||
| Software, algorithm | Data Analysis HT 12.0 | Sartorius | ||
| Other | Lipid Extract Bovine liver PC | Avanti Polar Lipids | 840055 | Powder |
| Other | Lipid Extract Bovine liver PE | Avanti Polar Lipids | 840026 | Powder |
| Other | Lipid Extract Porcine brain PS | Avanti Polar Lipids | 840032 | Powder |
| Other | Lipid Extract Bovine liver PI | Avanti Polar Lipids | 840042 | Powder |
| Other | Lipid Extract Porcine brain [PI(4,5)P2] | Avanti Polar Lipids | 840046 | Powder |
| Other | Lipid Extract Ovine wool cholesterol | Avanti Polar Lipids | 700000 | Powder |
| Other | Lipid Extract Chicken egg SM | Avanti Polar Lipids | 860061 | Powder |
| Other | Synthetic Lipid 16:0 Lissamine Rhod-PE | Avanti Polar Lipids | 810158 | Powder |
| Other | Synthetic Lipid 18:1 Biotinyl-PE | Avanti Polar Lipids | 870282 | Powder |
| Other | Synthetic Lipid 18:1 DGS-NTA [Ni-lipid] | Avanti Polar Lipids | 790404 | Powder |
| Other | Sartorius OctetRed96e instrument | Sartorius |
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Disulfide bridge-dependent dimerization triggers FGF2 membrane translocation into the extracellular space
eLife 12:RP88579.
https://doi.org/10.7554/eLife.88579.3
