Molecular and functional analysis of calcium binding by a cancer-linked calreticulin mutant
- Department of Microbiology and Immunology, University of Michigan Medical School, United States
- West African Centre for Cell Biology of Infectious Pathogens, Departments of Biochemistry, Cell and Molecular Biology, University of Ghana, Ghana
- Medical Laboratory Sciences, University of Ghana, Ghana
- Department of Pharmacology, University of Michigan Medical School, United States
Figures
Figure 1
Sequence alignments of the C-terminal residues of murine and human calreticulin (mCRT and hCRT), and the myeloproliferative neoplasm (MPN)-linked CRT mutants Ins5 and Del52.
Multiple sequence alignment using Clustal Omega was employed to compare C-domain sequences of mature mCRT, hCRT, Del52, and Ins5 proteins. Acidic residues (340–349) previously implicated in low-affinity calcium binding by mCRT that are shared between wild-type and both CRTDel52 and CRTIns5 are highlighted in green. Additional acidic residues shared just between wild-type CRT and CRTIns5 are shown in red. There are additional acidic residues unique to wild-type CRT. Mature protein numbering is used for the CRT sequences.
Figure 2
Purification of recombinant human wild-type CRT, CRTDel52, and C-domain truncation mutants.
(A) Representative chromatograms of recombinant GB1-and his-tagged human CRTWT (hCRTWT), CRTDel52, and the C-domain truncation mutants hCRT (1–351) and hCRT (1–339) purified by gel exclusion chromatography after expression in E. coli and initial purification using a nickel resin. (B) Representative Coomassie blue-stained SDS-PAGE gels of fractions corresponding to the monomer peak (those contained within the region indicated by the double-headed arrows on the chromatogram) of purified proteins that were used for calcium-binding measurements. This figure has Figure 2—source data 1, related to A, Figure 2—source data 2 and 3, related to B.
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Figure 2—source data 1
Chromatograms of purified recombinant CRT proteins.
- https://cdn.elifesciences.org/articles/109647/elife-109647-fig2-data1-v1.xlsx
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Figure 2—source data 2
Coomassie-stained SDS-PAGE gels.
- https://cdn.elifesciences.org/articles/109647/elife-109647-fig2-data2-v1.zip
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Figure 2—source data 3
Coomassie-stained SDS-PAGE gels.
- https://cdn.elifesciences.org/articles/109647/elife-109647-fig2-data3-v1.zip
Figure 3
Calcium binding to the low-affinity sites of purified human CRT proteins.
Low-affinity calcium binding by purified hCRTWT, CRTDel52, and the indicated truncation mutants was measured using isothermal titration calorimetry (ITC). (A–D) Representative isotherms and fitting curves of calcium binding to the low-affinity sites of the wild-type and mutant CRT proteins as indicated, measured using ITC. Prior to titration, the proteins were incubated with 50–100 µM CaCl2 to block the high-affinity calcium-binding sites. At 37°C, 10 mM CaCl2 was titrated against 100 µM protein. The heat change was measured for 25 injections, and the data were curve-fitted to calculate the dissociation constant, enthalpy, and binding stoichiometry using NanoAnalyze software. Data replicates and statistical analyses are specified in Table 1. This figure has Figure 3—source data 1, related to A–D.
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Figure 3—source data 1
Isothermal titration calorimetry measurements of calcium binding to CRT proteins.
- https://cdn.elifesciences.org/articles/109647/elife-109647-fig3-data1-v1.xlsx
Figure 4
Flow cytometry-based endoplasmic reticulum (ER) calcium measurements indicate similar ER calcium levels in CRT-KO HEK293T cells and those reconstituted with wild-type CRT and CRTDel52.
(A) CRISPR-Cas9 editing of human embryonic kidney (HEK) cells using lentiviral constructs with calreticulin-targeting gRNA (CRT-KO) or empty vector (HEK-Vec). CRT-KO HEK cells were subsequently reconstituted with wild-type human CRT, CRTDel52, or CRTDel52 with a C-terminal KDEL sequence as indicated. Representative blots showing expression levels of CRT in the indicated HEK cells. Cell lysates were subjected to SDS-PAGE followed by immunoblotting. Wild-type CRT, mutant CRT, and GAPDH were detected with anti-CRT (N), anti-CRT C-(mut), and anti-GAPDH antibodies, respectively. (B) Gating strategy for flow cytometry. HEK-Vec cells transiently transfected with the vector encoding the ratiometric GEM-CEPIA1er probe for 72 hr were used to assess ER calcium levels by flow cytometry based on the measurements of fluorescence emission signals for the Ca2+-bound or unbound probe in the Pacific Blue and Amcyan channels, respectively, following excitation with the 405 nm (violet) laser. Transfected cells were gated based on forward (FSC-A) and side scatter (SSC-A), then on 7AAD-negative (live) cells, and further on the cells positive for the calcium-bound and unbound probe signals. Representative dot plots of changes in fluorescence intensities in response to treatment with thapsigargin (Tg), a SERCA inhibitor, before (0 min) or after (10 min) treatment, are shown. (C) Representative histograms showing changes to Ca2+-bound and unbound signals at different time points following Tg treatment of cells. Mean fluorescence intensities (MFIs) for Ca2+-bound and unbound signals were used to calculate the bound/unbound ratios at different time points. (D and E) The basal free ER calcium signals were measured as bound/unbound MFI ratios from the corresponding histograms of CRT-KO or HEK-Vec cells (D, left panel) or CRT-KO cells reconstituted with the indicated CRT constructs (D, right panel). The fractional changes in ER calcium signals in response to Tg were measured in the indicated cells (E). The fractional change ratios were calculated as (R0–R10/R0)×100. Statistical significance was determined using GraphPad Prism and based on paired t-tests (left panels of D and E) or repeated-measures one-way ANOVAs with Tukey’s test (right panels of D and E). ns, not significant; ***p-value≤0.001. All measurements were undertaken in the presence of 2 mM extracellular Ca2+. Data shown in D and E are based on 7–15 independent experiments as indicated by the individual data points within the graphs. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This figure has Figure 4—source data 1 and 2, related to A, Figure 4—source data 3, related to D–E.
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Figure 4—source data 1
Immunoblots of CRT expression in HEK293T cells.
- https://cdn.elifesciences.org/articles/109647/elife-109647-fig4-data1-v1.zip
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Figure 4—source data 2
Immunoblots of CRT expression in HEK293T cells.
- https://cdn.elifesciences.org/articles/109647/elife-109647-fig4-data2-v1.zip
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Figure 4—source data 3
Flow cytometry-based endoplasmic reticulum (ER) calcium measurements in HEK293T cells.
- https://cdn.elifesciences.org/articles/109647/elife-109647-fig4-data3-v1.xlsx
Figure 5
Fluorimetry-based endoplasmic reticulum (ER) calcium measurements indicate similar ER calcium levels in CRT-KO HEK293T cells and those reconstituted with wild-type CRT and CRTDel52.
Human embryonic kidney (HEK) cells transiently transfected with a vector encoding the ratiometric GEM-CEPIA1er probe for 72 hr were used to assess ER calcium levels in the presence of 2 mM extracellular Ca2+. Fluorescence emission spectra were recorded following excitation at 390 nm. (A) Representative emission spectra show changes to Ca2+-bound (~460 nm) and unbound (~510 nm) peaks at different time points following Tg treatment of cells. The spectrum in blue was obtained with untransfected HEK cells. (B) Basal Ca2+-bound (~460 nm)/unbound (~510 nm) fluorescence intensity (FI) ratios were calculated for the indicated HEK cell lines before Tg addition. (C) The fractional changes to the bound (~460 nm)/unbound (~510 nm) fluorescence intensity ratios at 15 min relative to baseline were calculated as (R0–R15/R0)×100. Statistical significance was determined by repeated-measures one-way ANOVAs with Tukey’s test using GraphPad Prism. Data shown are based on four independent experiments, indicated by the individual data points within the graphs. ns, not significant. This figure has Figure 5—source data 1, related to B and C.
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Figure 5—source data 1
Spectrofluorimetric endoplasmic reticulum (ER) calcium measurements in HEK293T cells.
- https://cdn.elifesciences.org/articles/109647/elife-109647-fig5-data1-v1.xlsx
Figure 6 with 2 supplements
Calcium imaging studies indicate similar cytosolic calcium levels in the megakaryoblastic (MEG-01) cells with CRT-KO and those reconstituted with wild-type CRT or CRTDel52.
(A) CRISPR-Cas9 editing of MEG-01 cells using lentiviral constructs encoding calreticulin-targeting gRNA (CRT-KO). CRT-KO MEG-01 cells were subsequently reconstituted with viral constructs encoding wild-type human CRT or CRTDel52 or transduced with empty vector (Vec) as indicated in the figure panels. Representative immunoblots of CRT expression in the indicated cells detected by the anti-CRT(N) and anti-CRT(C-mut) antibodies. (B–F) Cells in 2 mM extracellular Ca2+ were loaded with the Fura-2/AM dye. Following excitation at 340 or 380 nm and recordings of emission at 510 nm, the emission ratios (340/380) are calculated. (B) Representative calcium imaging data from indicated cells in the presence of 2 mM extracellular Ca2+ and following ER calcium depletion with SERCA inhibitor, cyclopiazonic acid (CPA). (C and E) Basal cytosolic calcium levels are plotted as the mean 340/380 ratios. Data were normalized relative to the MEG-01 signals. (D and F) Mean fractional changes to cytosolic calcium levels calculated as (Rpeak–Rbaseline/Rbaseline)×100 and total calcium release determined as the area under the curve (AUC) are plotted. Data shown in C–F are based on four or five independent experiments with measurements averaged from 50 to 100 cells per experiment. Statistical analysis was performed using paired t-tests (C and D) or repeated-measures one-way ANOVAs with Tukey’s test (E and F). Statistical significance was determined based on p-value≤0.05 using GraphPad Prism. ns, not significant. This figure has Figure 6—source data 1 and 2 related to A, Figure 6—source data 3, related to C–D, and Figure 6—source data 4, related to E–F. This figure has two figure supplements: Figure 6—figure supplements 1 and 2.
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Figure 6—source data 1
Immunoblots of CRT expression in MEG-01 cells.
- https://cdn.elifesciences.org/articles/109647/elife-109647-fig6-data1-v1.zip
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Figure 6—source data 2
Immunoblots of CRT expression in MEG-01 cells.
- https://cdn.elifesciences.org/articles/109647/elife-109647-fig6-data2-v1.zip
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Figure 6—source data 3
Cytosolic calcium measurements in MEG-01 parental and CRT-KO cells.
- https://cdn.elifesciences.org/articles/109647/elife-109647-fig6-data3-v1.zip
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Figure 6—source data 4
Cytosolic calcium measurements in CRT-KO cells and those reconstituted with wild-type CRT and CRTDel52.
- https://cdn.elifesciences.org/articles/109647/elife-109647-fig6-data4-v1.zip
Figure 6—figure supplement 1
Imatinib treatment reduces p-STAT5 levels.
Representative immunoblots of STAT5 and phospho-STAT5 expression in the lysates of MEG-01 CRT-KO cells reconstituted with wild-type CRT or CRTDel52 or transduced with empty vector (Vec), as indicated, with or without imatinib treatment, detected by anti-stat5 and anti-phospho-stat5 antibodies. GAPDH is shown as loading control. This figure has Figure 6—figure supplement 1—source data 1 and 2.
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Figure 6—figure supplement 1—source data 1
Immunoblots of STAT5 and phospho-STAT5 in cells with or without imatinib treatment.
- https://cdn.elifesciences.org/articles/109647/elife-109647-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2
Immunoblots of STAT5 and phospho-STAT5 in cells with or without imatinib treatment.
- https://cdn.elifesciences.org/articles/109647/elife-109647-fig6-figsupp1-data2-v1.zip
Figure 6—figure supplement 2
Calcium imaging studies indicate similar cytosolic calcium levels in MEG-01 CRT-KO cells, and those reconstituted with wild-type CRT or CRTDel52 both with and without imatinib treatment.
Cells without (A) or with imatinib (IM) (B) treatment were loaded with Fura-2/AM dye in 2 mM extracellular Ca2+ followed by excitation at 340 or 380 nm and recording of emission signals at 510 nm. The basal cytosolic calcium levels before cyclopiazonic acid (CPA) addition are plotted as the mean ratios of emission signals following excitation at 340 or 380 nm (340/380) in cells without imatinib (A, left panel) or with imatinib treatment (B, left panel). Mean fractional changes to cytosolic calcium levels calculated as (Rpeak–Rbaseline/Rbaseline)×100 (Figure 6B) and total calcium release determined as the area under the curve (AUC), following CPA addition to cells without imatinib (A middle and right panels, respectively) or with imatinib (B, middle and right panels, respectively) treatment, are plotted. Data shown are based on six independent measurements, each including 40–70 cells. Repeated-measures one-way ANOVA with Tukey’s test was used for the statistical analyses. Statistical significance was determined based on p-value≤0.05 using GraphPad Prism. ns, not significant. This figure has Figure 6—figure supplement 2—source data 1, related to data replicates for panels A and B.
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Figure 6—figure supplement 2—source data 1
Cytosolic calcium measurements with and without imatinib treatment.
- https://cdn.elifesciences.org/articles/109647/elife-109647-fig6-figsupp2-data1-v1.zip
Figure 7 with 1 supplement
Calcium imaging studies indicate similar cytosolic calcium levels and store-operated calcium entry (SOCE) in MEG-01 cells with CRT-KO and those reconstituted with wild-type CRT or CRTDel52.
Cells were loaded with Fura-2/AM dye. Following excitation at 340 or 380 nm and recordings of emission at 510 nm, the emission ratios (340/380) were calculated. (A) Representative calcium imaging data from cells in the absence of extracellular calcium, followed by the addition of cyclopiazonic acid (CPA), and subsequent addition of extracellular calcium and CPA as indicated. (B and E) Basal cytosolic calcium levels are plotted as the mean 340/380 ratios prior to CPA addition. Data are normalized relative to the signals of cells transduced with empty vector (Vec). (C and F) Fractional changes to cytosolic calcium calculated as (Rpeak–Rbaseline/ Rbaseline)×100 (left panel) and total calcium release measured as the area under the curve (AUC) (right panel) are plotted for the indicated cells, following ER calcium depletion with CPA. (D and G) Fractional changes to cytosolic calcium following CPA and extracellular calcium additions (2 mM) are calculated as (Rpeak–Rbaseline/Rbaseline)×100, where Rbaseline and Rpeak were measured over 1 min intervals. Data shown are based on 8–10 independent experiments, and an average of over 20–100 cells in each experiment. Comparisons were performed by paired t-test (panels B–D) and one-way ANOVA (panels E–G) using GraphPad Prism. Statistical significance is based on a p-value≤0.05. ns, not significant, **p≤0.01. This figure has Figure 7—source data 1, related to B–D, Figure 7—source data 2, related to E–G. This figure has one supplement, Figure 7—figure supplement 1.
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Figure 7—source data 1
Cytosolic calcium measurements and store-operated calcium entry (SOCE) in parental and CRT-KO cells.
- https://cdn.elifesciences.org/articles/109647/elife-109647-fig7-data1-v1.zip
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Figure 7—source data 2
Cytosolic calcium measurements and store-operated calcium entry (SOCE) in CRT-KO cells and those reconstituted with wild-type CRT and CRTDel52.
- https://cdn.elifesciences.org/articles/109647/elife-109647-fig7-data2-v1.zip
Figure 7—figure supplement 1
Calcium imaging studies indicate similar cytosolic calcium levels and store-operated calcium entry (SOCE) in MEG-01 CRT-KO cells and those reconstituted with wild-type CRT or CRTDel52 both with and without imatinib treatment.
Cells without (top panels) or with imatinib (IM) (lower panels) treatment were loaded with Fura-2/AM dye, following excitation at 340 and 380 nm and emission at 510 nm. The ratio of the 340 nm/380 nm signals was calculated. Basal cytosolic calcium levels in the indicated cells without IM (A) left panel or with IM (C) left panel treatment in the absence of extracellular calcium was plotted as the ratios of 340 nm/380 nm signals. Fractional changes to cytosolic calcium and total calcium release measured as the area under the curve (AUC) in the indicated cells following ER calcium depletion with the cyclopiazonic acid (CPA) without IM (A) middle panels or with IM (C, middle panels) treatment. (B and D) Fractional changes to cytosolic calcium following ER calcium depletion with the CPA and subsequent addition of extracellular calcium in the presence of CPA in indicated cells without (B) or with IM (D) treatment. Data in middle panels of (A and C) are plotted as a fractional increase relative to baseline following CPA addition. SOCE in the indicated cells is plotted in panels (B and D) as the fractional increase in signal following the addition of 2 mM extracellular calcium in the presence of CPA. Data shown are based on six independent measurements, each with 30–60 cells. Comparisons were performed by one-way ANOVA using GraphPad Prism. Statistical significance is based on a p-value≤0.05. ns, not significant. This figure has Figure 7—figure supplement 1—source data 1, related to panels (A–D).
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Figure 7—figure supplement 1—source data 1
Cytosolic calcium measurements and store-operated calcium entry (SOCE) with and without imatinib treatment.
- https://cdn.elifesciences.org/articles/109647/elife-109647-fig7-figsupp1-data1-v1.zip
Figure 8
Transcriptional changes to the expression of genes encoding endoplasmic reticulum (ER) calcium-binding/regulating proteins in CRT-KO MEG-01 cells.
(A) RNA sequencing analysis was used to identify differentially expressed genes in MEG-01 CRT-KO cells in comparison to parental MEG-01 cells. Volcano plot shows significantly upregulated (blue) and downregulated (red) genes identified by RNA sequencing. Genes related to ER calcium binding and homeostasis have been highlighted and labeled. The differentially expressed genes were selected as those with a log2 fold-change of <−0.49 or >0.49 and p-values <0.05. (B) Heatmap shows the differential expression of genes encoding ER-resident calcium-binding proteins or ER calcium regulators that were identified by RNA sequencing in CRT-KO Meg-01 cells compared to parental cells (n=3 biological replicates). (C) Comparison of expression ratio of HSPA5, HSP90B1, PDIA3, and PDIA6 in CRT-KO MEG-01 cells (this study) or murine lung cancer cells (Tang et al., 2025), heterozygous CRTDel52 knock-in (Del52-KI) cells (Foßelteder et al., 2023) normalized to their expression in respective control cell lines expressing wild-type CRT. The normalized expression of various genes identified in each dataset was calculated as a ratio of expression in CRT-KO, CRTDel52 knock-in, or CRTDel52 cells relative to the expression in wild-type CRT-expressing cells. Multiple unpaired t-tests were used to determine statistical significance with the Benjamini-Hochberg FDR correction (p-adjusted)<0.05. (D–G) Left and middle panels: Relative expression of mRNA transcripts of HSPA5 (n=8–11), HSP90B1 (n=7), PDIA6 (n=6), and PDIA3 (n=6) genes in the indicated MEG-01 cells determined by RT-qPCR using specific gene primers. Relative gene expression was calculated by normalizing the target gene’s cycle threshold (Ct) to a single or multiple endogenous controls (ACTB, GAPDH, and HPRT1), which was used for comparison across different cell lines as indicated. Data show 6–11 independent runs with duplicate measurements for each run. Student’s t-test was used to determine the statistical significance between two groups while multiple group comparison was performed by one-way ANOVA using GraphPad Prism. Statistical significance is based on a p-value≤0.05. ns, not significant, *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. Right panels: Representative immunoblots of BiP, GRP94, PDIA6, and PDIA3 expression in the indicated cells detected by specific antibodies. This figure has Figure 8—source data 1, related to C, Figure 8—source data 2, related to D–G (left panels), and Figure 8—source data 3 and 4, related to D–G (right panels).
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Figure 8—source data 1
Comparison of expression ratio of HSPA5, HSP90B1, PDIA3, and PDIA6.
- https://cdn.elifesciences.org/articles/109647/elife-109647-fig8-data1-v1.xlsx
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Figure 8—source data 2
Relative expression of mRNA transcripts of HSPA5, HSP90B1, PDIA6, and PDIA3 genes.
- https://cdn.elifesciences.org/articles/109647/elife-109647-fig8-data2-v1.xlsx
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Figure 8—source data 3
Immunoblots of BiP, GRP94, PDIA6, and PDIA3 expression.
- https://cdn.elifesciences.org/articles/109647/elife-109647-fig8-data3-v1.zip
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Figure 8—source data 4
Immunoblots of BiP, GRP94, PDIA6, and PDIA3 expression.
- https://cdn.elifesciences.org/articles/109647/elife-109647-fig8-data4-v1.zip
Tables
Table 1
Calorimetric data on measurement of calcium binding to the low-affinity binding site of recombinant purified CRT.
| Recombinant CRT proteins/isothermal calorimetric measurement | ΔH (k/mol) | n (mol) | Dissociation constant – KD (µM) |
|---|---|---|---|
| CRTWT | 7.88±2.15 | 5.40±1.31 | 574.24±109.93 |
| CRT (1–351) | 79.82±32.55 | 2.59±1.56 | 444.34±139.99 |
| CRT (1–339) | Too low to reliably fit | ||
| CRTDel52 | 3.89±1.36 | 7.73±1.81 | 938.82±275.89 |
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Calorimetric data are represented as the mean ± SE. Data shown are averages from three to five independent replicates.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | Origami B(DE3) | Sigma-Aldrich | Ref 70836-M | Chemically competent |
| Cell line (human) | MEG-01 | ATCC | RRID:CVCL_0425 | This cell line was authenticated by Labcorp using STR genotyping |
| Cell line (human) | HEK293T | ATCC | RRID:CVCL_0063 | This cell line was authenticated by Labcorp using STR genotyping |
| Antibody | Anti-CRT (N) (rabbit monoclonal) | Cell Signaling Technology | CST #12238;RRID:AB_2688013 | 1:5000 |
| Antibody | Anti-CRT (C-mut) | PMCID:PMC8085772 | 1:10,000 | |
| Antibody | Anti-GRP78/BiP (rabbit polyclonal) | Proteintech | 11587–1-AP;RRID:AB_2119855 | 1:10,000 |
| Antibody | Anti-GRP94 (rabbit polyclonal) | Proteintech | 14700–1-AP;RRID:AB_2233347 | 1:10,000 |
| Antibody | Anti-ERp57 or PDIA3 (rabbit polyclonal) | Proteintech | 15967–1-AP;RRID:AB_2236784 | 1:10,000 |
| Antibody | Anti-PDIA6 (rabbit polyclonal) | Proteintech | 18233–1-AP;RRID:AB_10805765 | 1:20,000 |
| Antibody | Anti-STAT5 (rabbit monoclonal) | Cell Signaling Technology | CST #94205;RRID:AB_2737403 | 1:1000 |
| Antibody | Anti-phospho-STAT5 (rabbit monoclonal) | Cell Signaling Technology | CST # 9359;RRID:AB_823649 | 1:1000 |
| Antibody | Anti-vinculin | Cell Signaling Technology | CST 13901;RRID:AB_2728768 | 1:20,000 |
| Antibody | Anti-GAPDH | Cell Signaling Technology | CST 2118;RRID:AB_561053 | 1:20,000 |
| Recombinant DNA reagent | pCIS-GEM-CEPIA1er (plasmid) | Addgene | RRID:Addgene_58217 | |
| Recombinant DNA reagent | psPAX2 (plasmid) | Addgene | RRID:Addgene_12260 | |
| Recombinant DNA reagent | pMD2.G | Addgene | RRID:Addgene_12259 | |
| Recombinant DNA reagent | pMSCV (plasmid) | Addgene | RRID:Addgene 75085 | |
| recombinant DNA reagent | ZsGreen1 (pLVX-IRES-Puro) | Addgene | RRID:Addgene_180278 | |
| Recombinant DNA reagent | pLentiCRISPRv2-BLAST (pLB) | Addgene | RRID:Addgene_98293 | |
| Recombinant DNA reagent | pSIP-CALRWT (plasmid) | This paper | Wild-type calreticulin (CALRWT) cDNA cloned in pSIP-ZsGreen (pLVX-SFFV-IRES-Puro) vector at EcoRI and NotI sites | |
| Recombinant DNA reagent | pSIP-CALRDel52 (plasmid) | This paper | Mutant calreticulin (CALRDel52) cDNA cloned in pSIP-ZsGreen (pLVX-SFFV-IRES-Puro) vector at EcoRI and NotI sites | |
| Recombinant DNA reagent | pSIP-CALRDel52+KDEL (plasmid) | This paper | Mutant calreticulin+KDEL (CALRDel52+KDEL) cDNA cloned in pSIP-ZsGreen (pLVX-SFFV-IRES-Puro) vector at EcoRI and NotI sites | |
| Recombinant DNA reagent | pGB1-CALRWT (plasmid) | This paper | B1 domain of streptococcal protein G (GB1) (Huth et al., 1997) and hexa-histidine tag using the pGB1 vector via ligation-independent cloning, as described earlier (Del Cid et al., 2010; DelProposto et al., 2009) | |
| Recombinant DNA reagent | pGB1-CALR(1-351) (plasmid) | This paper | B1 domain of streptococcal protein G (GB1) (Huth et al., 1997) and hexa-histidine tag using the pGB1 vector via ligation-independent cloning, as described earlier (Del Cid et al., 2010; DelProposto et al., 2009) with quickchange site mutagenesis | |
| Recombinant DNA reagent | pGB1-CALR(1-339) (plasmid) | This paper | B1 domain of streptococcal protein G (GB1) (Huth et al., 1997) and hexa-histidine tag using the pGB1 vector via ligation-independent cloning, as described earlier (Del Cid et al., 2010; DelProposto et al., 2009) with quickchange site mutagenesis | |
| Sequence-based reagent | ACTB (Primer) | Real-time primers | VHPS-110 | |
| Sequence-based reagent | GAPDH (Primer) | Real-time primers | VHPS-3541 | |
| Sequence-based reagent | HPRT1 (Primer) | Real-time primers | VHPS-4263 | |
| Commercial assay or kit | Pierce ECL western blotting substrate | Thermo Fisher Scientific | Cat #32106 | |
| Commercial assay or kit | Pierce BCA protein quantification assay | Thermo Fisher Scientific | Cat #23225 | |
| Commercial assay or kit | RNeasy Plus Mini Kit | QIAGEN | Ref 74136 | |
| Commercial assay or kit | Superscript III First-Strand Synthesis | Thermo Fisher Scientific | Ref 18080051 | |
| Commercial assay or kit | SYBR Green PCR Master Mix | Applied Biosystems | Ref 4309155 | |
| Commercial assay or kit | Lipofectamine LTX with PLUS reagent | Invitrogen | Ref 15338100 | |
| Commercial assay or kit | QuickChange II XL Site-Directed Mutagenesis | Agilent Technologies | Cat #200522 | |
| Chemical compound, drug | Fura-2 LR AM | ION Biosciences | 1061E | |
| Chemical compound, drug | Thapsigargin | Enzo Life Sciences | BML-PE180-0001 | |
| Chemical compound, drug | Cyclopiazonic acid | Cayman Chemical | 11326 | |
| Chemical compound, drug | Pluronic F-127 | Sigma-Aldrich | P2443 | |
| Chemical compound, drug | Imatinib | Sigma-Aldrich | CDS022173 | |
| Software, algorithm | GraphPad Prism | Dotmatics | RRID:SCR_002798 | |
| Software, algorithm | Microsoft Excel | Microsoft | RRID:SCR_016137 |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/109647/elife-109647-mdarchecklist1-v1.docx
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Supplementary file 1
List of primers used in this study for cloning, site-directed mutagenesis, and qPCR.
- https://cdn.elifesciences.org/articles/109647/elife-109647-supp1-v1.docx
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Molecular and functional analysis of calcium binding by a cancer-linked calreticulin mutant
eLife 15:RP109647.
https://doi.org/10.7554/eLife.109647.3
