PP2A/B55α substrate recruitment as defined by the retinoblastoma-related protein p107
- Institute for Cancer Research, Fox Chase Cancer Center, United States
- Fels Institute for Cancer Research and Molecular Biology, Temple University Lewis Katz School of Medicine, United States
- Department of Chemistry and Biochemistry, University of Arizona, United States
- Department of Biochemistry and Cell Biology, Hitchcock Medical Center at Dartmouth, United States
- Department Cell Biology, UConn Health, United States
- Department Molecular Biology and Biophysics, UConn Health, United States
Figures
Figure 1 with 1 supplement
The intrinsically disordered spacer region of p107 contains three highly conserved regions (R1, R2, and R3), of which R1 is required for B55α binding and R2 enhances the binding interaction as determined via mutational analysis and NMR.
(A) The spacer and the C-terminus of p107 are intrinsically disordered (IUPred2a web interface, Sievers et al., 2011). (B) Clustal W alignment of conserved amino acid sequences of the p107 spacer …
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Figure 1—source data 1
Uncropped replicates of western blot images and PVDF membranes stained with Coomassie Blue used to quantitate B55α binding to conserved regions in the p107 spacer (top bar graph).
B55α signal was normalized to the GST-fusion protein signal (selected bands are marked with dashed white boxes and corrected for background from a band-less identical area).
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig1-data1-v2.zip
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Figure 1—source data 2
Uncropped replicates of western blot images and PVDF membranes stained with Coomassie Blue or Ponceau S used to quantitate binding to B55α of conserved regions in the p107 R1–R2 construct (top bar graph) and R/K point mutant variants of p107 R1–R2 (lower bar graph).
Only the R/K mutants labeled blue were included in the quantitation.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig1-data2-v2.zip
Figure 1—figure supplement 1
Clustal W alignment of conserved amino acid sequences of the p107 spacer from different species.
(A) Complete Clustal W alignment of conserved amino acid sequences of the p107 spacer from different species. Three highly conserved regions within the spacer were identified, which are highlighted …
Figure 2 with 1 supplement
Mutation of highly conserved residues on the β-propeller top of B55α have substrate-specific effects on binding, supporting the notion that substrates contact different surfaces on B55α.
(A) ConSurf depiction of PP2A/B55α mapping amino acid conservation (where amino acids are color-coded by conservation). (B) Electrostatic predictions mapped to the surface of the PP2A/B55α structure …
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Figure 2—source data 1
Upper, middle, and lower western blot membranes for Figure 2D (replicate 1).
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig2-data1-v2.zip
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Figure 2—source data 2
Western blot membranes for replicates 1–3.
All replicates were used for the quantitation shown in Figure 2E. The legends indicate the B55α variants used in this set of replicates. Relevant proteins and IgG (in the IP membranes) are indicated.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig2-data2-v2.zip
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Figure 2—source data 3
Western blot membranes for replicates 1–2 used for the quantitation shown in Figure 2E.
The legend indicates the B55α variants used in this set of replicates. Relevant proteins and IgG (in the IP membranes) are indicated.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig2-data3-v2.zip
Figure 2—figure supplement 1
Effect of B55α mutations on p107, pRB and KSR1 binding.
(A) Structure of the PP2A/B55α holoenzyme (PDB:3DW8). (B) Table depicting complete list of Myc-tagged B55α mutants generated, as well as summarizing the effect on binding to p107 and PP2A/A or …
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Figure 2—figure supplement 1—source data 1
Western blot membranes for replicates used for the quantitation of Flag-pRB:Myc-B55α binding ratios shown in Figure 2—figure supplement 1 (middle).
The legends indicate the B55α variants used in this set of replicates. Relevant proteins and IgG (in the IP membranes) are indicated.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig2-figsupp1-data1-v2.zip
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Figure 2—figure supplement 1—source data 2
Western blot membranes for replicates used for the quantitation of Flag-pRB:Myc-B55α binding ratios shown in Figure 2—figure supplement 1 (middle).
The legends indicate the B55α variants used in this set of replicates. Relevant proteins and IgG (in the IP membranes) are indicated.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig2-figsupp1-data2-v2.zip
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Figure 2—figure supplement 1—source data 3
Western blot membranes for replicates used for the quantitation of Flag-pRB:Myc-B55α binding ratios shown in Figure 2—figure supplement 1 (middle).
The legends indicate the B55α variants used in this set of replicates. Relevant proteins and IgG (in the IP membranes) are indicated.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig2-figsupp1-data3-v2.zip
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Figure 2—figure supplement 1—source data 4
Upper, middle, and lower western blot membranes for Figure 2—figure supplement 1, bottom.
Boxes indicate approximate area shown in the figure. Relevant proteins are indicated.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig2-figsupp1-data4-v2.zip
Figure 3 with 1 supplement
Combination of in vitro enzymatic assays and mass spectrometry identified S615 on R1 of p107 as the major site of PP2A/B55α-mediated dephosphorylation.
(A) Dephosphorylation of GST-p107 R1R2 was performed using approximately 10 ng purified PP2A/B55α. The indicated time points were collected and samples were resolved via SDS-PAGE. Proteins were …
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Figure 3—source data 1
Uncropped blots and Coomassie-stained gels for Figure 3A-C.
Images in Figure 3A were generated from the boxed regions in each PVDF membrane. Comparable experiments are shown in Figure 4. Images in Figure 3B were generated from the boxed regions in replicate 1. Replicates 1–3 show comparable dephosphorylation of WT and MT p107 R1R2 by Coomassie Blue staining. The gel image in Figure 3C was generated form representative replicate 1, and the bands cut out for mass spectrometry are boxed.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig3-data1-v2.zip
Figure 3—figure supplement 1
Experiments performed to determine optimal GST-p107 R1R2 phosphorylation parameters using purified cyclin A/CDK2.
(A) Phosphorylation of purified GST-p107 R1R2 was performed using 0.25 μg recombinant cyclin A/CDK2 and 5 μCi γ–32P. The indicated time points were collected and samples were resolved via SDS-PAGE. …
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Figure 3—figure supplement 1—source data 1
Uncropped Coomassie-stained gel, Phosphorimager exposure and western blots for Figure 3—figure supplement 1.
Dashed boxes correspond to the areas shown in Figure 3—figure supplement 1 (top). The 7.5’ sample was loaded after the 15” sample in error. The lane was omitted in the figure for clarity and the omitted lane marked with an asterisk.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig3-figsupp1-data1-v2.zip
Figure 4
Residues critical for B55α/PP2A binding to p107 are critical for p107 spacer dephosphorylation.
(A) Approximately 10 ng of purified PP2A/B55α was used to dephosphorylate wildtype GST-p107 R1R2 or mutant constructs (GST-p107 R621A K623A and GST-p107 RxK K657A R659A) in a time-course experiment. …
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Figure 4—source data 1
Uncropped Coomassie-stained gels and blots for Figure 4A and B.
Images in Figure 4A were generated from the boxed regions in replicate 1 PVDF membrane and the Coomassie Blue-stained gel. Quantifications shown in Figure 4A of the ‘phospho’-p107 band were obtained from Coomassie Blue-stained gel replicates 1–3. Images in Figure 4B were generated from the boxed regions in replicate 1. A comparable experiment using an R1 peptide (R1-627TDS-AAA) variant that binds B55α (Figure 5B) showed delayed phosphorylation as R1, while R1-R621A and R2, which do not bind B55α (Figure 5A and C), did not inhibit dephosphorylation.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig4-data1-v2.zip
Figure 5 with 1 supplement
Identification of critical residues within the central 9-mer stretch of the ‘R1’ region of p107 for binding to B55α/PP2A (SPxxHxRVxxV).
(A) PP2A/B55α purified from 293T-Flag-B55α cells was preincubated with synthetic p107 peptides and then used in pull-down assays with GST-p107 R1R2 constructs. Proteins were resolved via SDS-PAGE …
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Figure 5—source data 1
Uncropped upper membrane (anti-B55α) and lower membrane (anti-GST) western blots for Figure 5A and B.
Images in Figure 4A and B were generated from the boxed regions in replicate 1 PVDF membranes. B55α band intensities were normalized to the corresponding full-length GST-R1R2 band intensities.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig5-data1-v2.zip
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Figure 5—source data 2
Uncropped upper membrane (anti-B55α) and lower membrane (anti-GST) western blots for Figure 5B, C (three replicates) were used to quantitate peptide competition of B55α binding to GST-R1R2.
B55α band intensities were normalized to the corresponding full-length GST-R1R2 band intensities.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig5-data2-v2.zip
Figure 5—figure supplement 1
Identifying esential rediues in the R1 region of the p107 spacer that affect binding to B55α, using peptide competition and GST puldown assays.
(A) Representative pulldowns were performed using purified PP2A/B55α (PP2A) and GST-p107 R1R2 with preincubations using mutant p107-derived synthetic peptides, phosphopeptides, and control peptides, …
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Figure 5—figure supplement 1—source data 1
Uncropped western blot images for panel B.
All the replicates for panel A are shown in Figure 5—source data 2.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig5-figsupp1-data1-v2.zip
Figure 6 with 1 supplement
p107 R1 interaction with the B55α holoenzyme in cells depends on sites required for binding and dephosphorylation in vitro.
(A) GFP-p107 R1 wildtype and mutant constructs were co-transfected with Myc-B55α wildtype and B55α-D197K mutant constructs into 293T cells and used for IPs with anti-GFP agarose conjugate. Input …
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Figure 6—source data 1
Uncropped upper membranes for Figure 6A and replicate experiments of endogenous B55α interaction with wildtype, but not mutant, GFP-R1-SLiM.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig6-data1-v2.zip
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Figure 6—source data 2
Mass spectrometry dataset used to generate the volcano plot shown in Figure 6.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig6-data2-v2.xlsx
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Figure 6—source data 3
Uncropped upper and lower membranes for Figure 6C and D (representative replicate 1 was selected for the C and D panels).
In (C), B55α-mediated dephosphorylation was quantitated using the pCDK substrate vs. Flag-p107 signal using the corresponding bands in the dashed boxes.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig6-data3-v2.zip
Figure 6—figure supplement 1
U-2 OS cells were co-transfected with the indicated plasmids, lysates were immunoprecipitated with anti-Flag agarose conjugate, and the indicated co-immunoprecipitated proteins were detected by western blot analyses.
(Right) Quantification of Flag-p107/HA-E2F4 rations is shown.
Figure 7 with 2 supplements
A computational model of the p107 phosphopeptide (613–622) binding B55α and the active site of PP2A/C is consistent with p107 spacer contacts to B55α as determined by NMR spectroscopy.
(A) The ‘two-fragment’ model depicting B55α and PP2A/C with two modeled peptide fragments. Distances between the OE1/OE2 atoms of Glu and the NH1/NH2 atoms of Arg are shown in center. (B) A closer …
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Figure 7—source data 1
PyMOL session source data for Figure 7A.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig7-data1-v2.zip
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Figure 7—source data 2
PyMOL session source data for Figure 7B.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig7-data2-v2.zip
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Figure 7—source data 3
PyMOL session source data for Figure 7C and D.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig7-data3-v2.zip
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Figure 7—source data 4
PyMOL session source data for Figure 7E.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig7-data4-v2.zip
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Figure 7—source data 5
PyMOL session source data for Figure 7F.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig7-data5-v2.zip
Figure 7—figure supplement 1
Step details to generate a computational model of the p107 phosphopeptide (613-622) binding B55α and the active site of PP2A/C.
(A) Density plot of distances between OE1/2 of Glu and NH1/2 of Arg in 520 peptide structures with the consensus sequence EPXXXPR. The red dot is the distance between OE1/2 of Glu and NH1/2 of Arg …
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Figure 7—figure supplement 1—source data 1
Distances between OE1/2 and NH1/2 for 520 peptide structures with the consensus sequence EPXXXPR retrieved from PDB.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig7-figsupp1-data1-v2.txt
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Figure 7—figure supplement 1—source data 2
R script file to generate density plots for Figure 7—figure supplement 1A and B.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig7-figsupp1-data2-v2.txt
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Figure 7—figure supplement 1—source data 3
PyMOL session source data for Figure 7—figure supplement 1B.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig7-figsupp1-data3-v2.zip
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Figure 7—figure supplement 1—source data 4
Distances of OE1/2 and NH1/2 between each peptide and two reference fragments for Figure 7—figure supplement 1C.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig7-figsupp1-data4-v2.zip
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Figure 7—figure supplement 1—source data 5
PyMOL session source data for Figure 7—figure supplement 1D.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig7-figsupp1-data5-v2.zip
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Figure 7—figure supplement 1—source data 6
PyMOL session source data for Figure 7—figure supplement 1E.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig7-figsupp1-data6-v2.zip
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Figure 7—figure supplement 1—source code 1
Model code.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig7-figsupp1-code1-v2.zip
Figure 7—figure supplement 2
PP2A/A scaffold flexibility upon binding to the catalytic subunit and B subunits of the four distinct holoenzymes.
(A) Comparison of the flexibility of the PP2A/A scaffold upon binding to the catalytic subunit and B subunits of the four distinct holoenzymes. B55/B is colored red, B56/B′ is colored yellow, …
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Figure 7—figure supplement 2—source data 1
PyMOL session source data for Figure 7—figure supplement 2A.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig7-figsupp2-data1-v2.txt
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Figure 7—figure supplement 2—source data 2
PyMOL session source data for Figure 7—figure supplement 2B.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig7-figsupp2-data2-v2.txt
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Figure 7—figure supplement 2—source data 3
Scaffold variation source data without regulatory subunit for Figure 7—figure supplement 2C.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig7-figsupp2-data3-v2.zip
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Figure 7—figure supplement 2—source data 4
Scaffold variation source data with B56 subunits for Figure 7—figure supplement 2D.
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig7-figsupp2-data4-v2.zip
Figure 8 with 1 supplement
A derived p107 -pSPxxHxRVxxV- short linear motif (SLiM) is conserved in other substrates and functional validated in TAU.
(A) Schematic of our proposed hypothetical consensus groove SLiM, p[ST]-P-x(4,10)-[RK]-V-x-x-[VI]-R, for TAU, MAP2, and the conserved p107 family member, p130, each of which contain residues that …
Figure 8—figure supplement 1
Degenerate short linear motif (SLiM) search in the proteome vs. datasets enriched for B55 interactor and potential substrates.
The bar graph represents the percent of proteins with a degenerate version of the SLiM, [ST]-P-x(4,10)-[RK]-[VIL]-x(2)-[VILM] in the proteome and in a dataset of B55α interactors (Hertz et al., 2016)…
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Figure 8—figure supplement 1—source data 1
Table of proteins containing the [ST]-P-x(4,10)-[RK]-[VIL]-x(2)-[VILM] sequence in a dataset of B55α interactors (Hertz et al., 2016) and potential in vitro B55α mitotic substrates (Kruse et al., 2020).
- https://cdn.elifesciences.org/articles/63181/elife-63181-fig8-figsupp1-data1-v2.xlsx
Author response image 1
Pull down assays with the indicated fusion proteins from HEK-293 lysates were performed and binding of B55a was determined by western blot analysis.
Two replicates of the experiment showed comparable results.
Author response image 2
GST-R1R2 was phosphorylated with Cyclin A/CDK2 where indicated as described in the Manuscript.
B55a binding to GST-R1R2 was determined in the presence or absence of OA to prevent dephosphorylation. Proteins were resolved via SDS-PAGE and detected by western blotting using anti-B55α and GST …
Author response image 3
HEK-293 cells with doxicycline (Dox) inducible WT B55a and B55aD197K stable transgenes where treated with Dox for the indicated times and expression of indicated proteins and phospho pRB were determined by western blot analysis.
Author response image 4
BJ-TERT fibroblasts with doxicycline (Dox) inducible WT B55a and B55aD197K stable transgenes where grown to confluency treated with Dox for for 48 h, serum starved for 24 hours and then restimulated with Medium conaning 10% FBS for the indicated times.
Expression of indicated proteins and phospho pRB was determined by western blot analysis.
Author response image 5
Pull down assays with the indicated fusion proteins from HEK-293 lysates were performed and binding of B55a and Cyclin A was determined by western blot analysis.
Two replicates of the experiment showed comparable results.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | CDK2(rabbit polyclonal) | Santa Cruz | sc-163 | WB(1:1000) |
| Antibody | Cyclin A(mouse monoclonal) | Santa Cruz | sc-271682 | WB(1:1000) |
| Antibody | Flag(mouse monoclonal) | Sigma | A8592-.2MG | WB(1:2500, 1:10,000) |
| Antibody | Flag(mouse monoclonal) | GenScript | A00187 | IP (1 µg/mL) |
| Antibody | GFP(rabbit monoclonal) | CST | 2956S | WB(1:1000) |
| Antibody | GST(mouse monoclonal) | Santa Cruz | sc-138 | WB(1:1000) |
| Antibody | PP2A Aβ subunit(goat polyclonal) | Santa Cruz | sc-6113 | WB(1:2000) |
| Antibody | PP2A B subunit(100C1)(rabbit monoclonal) | CST | 2290S | WB(1:2000) |
| Antibody | PP2A C subunit (1D6) (mouse monoclonal) | Millipore | 05-421 | WB(1:5000) |
| Antibody | Phospho-CDK Substrate Motif(rabbit monoclonal) | CST | 9477S | WB(1:1000) |
| Antibody | ECL Rabbit IgG, HRP-linked whole Ab (from donkey) | GE Healthcare | NA934V | WB/secondary antibody(1:10,000) |
| Antibody | HA (12CA5)(mouse monoclonal) | Roche/Sigma | 11583816001 | WB(1:500) |
| Antibody | ECL Mouse IgG, HRP-linked whole Ab (from donkey) | GE Healthcare | NA931V | WB/secondary antibody(1:10,000) |
| Antibody | Mouse anti-goat IgG-HRP | Santa Cruz | sc-2354 | WB/secondary antibody(1:10,000) |
| Antibody | Monoclonal ANTI-FLAG M2 antibody produced in mouse, ANTI-FLAG M2 Affinity Agarose Gel | Sigma | A2220 | IP (10 μL) |
| Antibody | Anti-c-Myc Agarose Affinity Gel antibody produced in rabbit (polyclonal) | Sigma | A7470 | IP (10 μL) |
| Antibody | GFP-Trap agarose beads | Chromotek | gta-10 | IP (5 μL) |
| Peptide, recombinant protein | Synthetic peptides for competition assays | Biomatik | Custom | Sequence variant described in this paper |
| Peptide, recombinant protein | DYKDDDDK peptide | GenScript | RP10586 | |
| Peptide, recombinant protein | Recombinant cyclin A/CDK2 | Thermo Fisher | PV3267 | |
| Peptide, recombinant protein | Purified recombinant B55α/PP2A holoenzyme | Zhao et al., 2019 | Trimeric Flag-B55α/PP2A holoenzyme purified from 293T cells. | |
| Strain, strain background (Escherichia coli) | BL21-Gold (DE3) cells | Agilent | 230132 | To generate GST-Fusion proteins |
| Cell line (Homo sapiens) | 293T cells | ATCC | CRL-3216 | Transient transfections and source of cell lysates |
| Cell line (H. sapiens) | U-2 OS cells | ATCC | HTB-96 | Transient transfections |
| Cell line (H. sapiens) | Expi293F cells | Thermo Fisher | A14528 | Purification of recombinant B55α |
| Transfected construct (human) | Flag-B55α 293T cells | Zhao et al., 2019 | 293T cells stably transfected with pCPP-Flag-B55α and selected with puromycin | |
| Commercial assay or kit | (AminoLink Plus Immobilization Kit) | Thermo Fisher Scientific | 44894 | |
| Commercial assay or kit | Ser/Thr phosphatase assay kit | EMD Millipore | 17-127 | |
| Commercial assay or kit | QuikChange II | Agilent | 200521 | |
| Recombinant DNA reagent | pBOS GFP-H2B plasmid | Kanda et al., 1998 | ||
| Recombinant DNA reagent | pcDNA3.4-K-GFP-RP1B | This paper | His6-green fluorescent protein-tag, a TEV cleavage | |
| Recombinant DNA reagent | pTHMT | Peti and Page, Protein Expr. Purif. 51, 1–10 (2007) | N-terminal His6-Maltose Binding Protein (MBP)-tag, a TEV cleavage | |
| Recombinant DNA reagent | pCPP-Flag-B55α | Zhao et al., 2019 | ||
| Recombinant DNA reagent | pMSCV-puro-Myc-B55α | Jayadeva et al., 2010 | ||
| Recombinant DNA reagent | pMSCV-puro-Myc-B55α variants | This paper | Generated by site-directed mutagenesis (primer sequences provided in Appendix table) | |
| Recombinant DNA reagent | pCDNA5/FRT/TO-GFP-p107-R1 | This paper | Inserting wild-type or mutant p107-R1 gBlock Gene Fragments (IDT) into the pCDNA5/FRT/TO-GFP vector via the BamHI/Not sites | |
| Recombinant DNA reagent | pGEX-2T-p107 spacer | Jayadeva et al., 2010 | ||
| Recombinant DNA reagent | pGEX-2T-p107 spacer variants | This paper | Generated by site-directed mutagenesis (primer sequences provided in Appendix table) | |
| Recombinant DNA reagent | pCDNA5/FRT/TO-GFP-p107-R1 | This paper | Inserting a gblock in pCDNA5/FRT/TO-GFP containing 4 copies of R1 | |
| Recombinant DNA reagent | pCDNA5/FRT/TO-GFP-p107-R1-H/AxR/AV/AxxV/A | This paper | Inserting a gblock in pCDNA5/FRT/TO-GFP containing four mutant copies of R1. (gblock sequences provided in Appendix table) | |
| Recombinant DNA reagent | pSG5-puro-Flag-p107 | Kurimchak et al., 2013 | ||
| Recombinant DNA reagent | pCMV-Flag-p107 | Voorhoeve et al., 1999 | ||
| Recombinant DNA reagent | pCMV-HA-E2F4 | Ginsberg et al., 1994 | ||
| Recombinant DNA reagent | pRC-cyclin E | Addgene | #8963 | |
| Software, algorithm | ConSurf | ConSurfAshkenazy et al., 2010 | ||
| Software, algorithm | ImageJ software | |||
| Software, algorithm | FlowJo v10 software | BD Biosciences | v10.8 | |
| Software, algorithm | Clustal Omega | Sievers et al., 2011 |
