rabbit anti stim1  (Cell Signaling Technology Inc)

Journal: Cell Death Discovery

Article Title: Lysine attenuates acute lung injury by restoring α-tubulin acetylation and ciliary activity

doi: 10.1038/s41420-026-03025-x

Figure Lengend Snippet: Calcium image analysis in PQ-injured A549 cells compared to normal cells ( A ) and PQ-injured A549 cells w/wo lysine supplementation ( B ) with thapsigargin (TG) or ionomycin (IONO) stimulation. Mean ± SEM. C NFAT luciferase expression in A549 cells treated w/wo 800 μM PQ, together with 0-, 1-, 2.5-, or 5-fold lysine as in the culture medium as indicated, for 24 h. Mean ± SD, *** P < 0.001; Two-Way ANOVA. n = 3. NS not significant. D WB analysis of E-Cadherin, ZO-1, EPCAM and acetyl-α-Tubulin in PQ-injured A549 cells treated w/wo 5 μM SKF-96365 (SKF). E Co-IP analysis of STIM1 association with ORAI1 or TRPC1 in PQ-injured A549 cells with indicated concentration, together w/wo 5 mM lysine. F Immunofluorescence analysis of Myc and ARL13B in PQ-injured TRPC1-Myc stably expressed A549 cells w/wo lysine supplementation. Scale bar, 10 μm.

Article Snippet: Specific antibodies were used for STIM1 (Cell Signaling Technology, 5668, 1:3000), ORAI1 (Santa Cruz, sc-377281, 1:500), and TRPC1 (Proteintech, 19482, 1:2000).

Techniques: Luciferase, Expressing, Co-Immunoprecipitation Assay, Concentration Assay, Immunofluorescence, Stable Transfection

Journal: Cell Death Discovery

Article Title: Lysine attenuates acute lung injury by restoring α-tubulin acetylation and ciliary activity

doi: 10.1038/s41420-026-03025-x

Figure Lengend Snippet: A GSEA analysis uncovered top-ranked molecular functions enhanced in PQ-injured lung tissues with lysine supplementation compared to the untreated group. B KEGG enrichment of the top 20 upregulated pathways in PQ-injured lung tissues with lysine supplementation compared to the untreated group. C Quantification of NADP and NADPH (left) or NAD and NADH (right) in PQ-injured A549 cells w/wo lysine supplementation. D ELISA analysis of acetyl coenzyme A (Acetyl-CoA) in PQ-injured A549 cells w/wo lysine supplementation. E WB analysis of E-Cadherin and acetyl-α-Tubulin in PQ-injured A549 cells treated w/wo 1 mM sodium acetate (SA). F WB analysis of ZO-1, E-Cadherin, acetyl-α-Tubulin and EPCAM in PQ-injured A549 cells w/wo lysine supplementation, together w/wo 0.5 μM GM-90257 (GM). G Calcium image analysis in PQ-injured A549 cells w/wo lysine supplementation, together w/wo 0.5 μM GM-90257 treatment. The cells were transiently stimulated with thapsigargin (TG) or ionomycin (IONO). Mean ± SEM. H Co-IP analysis of STIM1 association with TRPC1 in PQ-injured A549 cells w/wo lysine supplementation, together w/wo GM-90257 treatment. Immunofluorescence analysis of ARL13B, SFTPC, and HOPX in A549 cells ( I ) or normal lung tissues ( J ). Scale bar, 10 μm. Mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001; Two-Way ANOVA. NS not significant.

Article Snippet: Specific antibodies were used for STIM1 (Cell Signaling Technology, 5668, 1:3000), ORAI1 (Santa Cruz, sc-377281, 1:500), and TRPC1 (Proteintech, 19482, 1:2000).

Techniques: Enzyme-linked Immunosorbent Assay, Co-Immunoprecipitation Assay, Immunofluorescence

Journal: The EMBO Journal

Article Title: STIM1-containing contact sites promote direct calcium flux from the endoplasmic reticulum to mitochondria

doi: 10.1038/s44318-026-00700-8

Figure Lengend Snippet: ( A ) STIM1-GFP or GFP (empty GFP vector) expression was induced with 1 μg/ml doxycycline for 22 h in STIM1-KO HEK293 cells. Immunoprecipitation of GFP-tagged proteins was performed using 1 mg of whole cell lysate (WCL), and co-precipitated proteins were analyzed by immunoblotting. For detecting VDAC1 and VDAC3, two different specific antibodies were tested, yielding the same result for both proteins. The antibodies used for VDAC1 were sc-390996 (Santa Cruz Biotechnology) and 10866-1-AP (Proteintech), while those for VDAC3 were PA5-51156 (ThermoFisher Scientific) and 55260-1-AP (Proteintech). As a positive control, 3 μg WCL was loaded (WCL lane). Blots are representative of 3 biological replicates. Levels of immunoprecipitated GFP or STIM1-GFP were analyzed as a loading control. ( B ) Total levels of GRP75 and VDAC proteins in WCL were evaluated by immunoblotting (30 μg protein/lane). ( C ) WCL (2.5 mg) was incubated with the anti-STIM1 antibody followed by Dynabeads™. Co-precipitation of GRP75 with endogenous STIM1 was evaluated by immunoblotting with a specific antibody (upper panel), which also detected STIM1 non-specifically due to the high amount of immunoprecipitated protein. As a positive control, 1 μg WCL was loaded. As a negative control, normal rabbit IgG was used instead of anti-STIM1 antibody (Ig lane). Blots are representative of 3 technical replicates from 2 biological replicates. Immunoprecipitated STIM1 levels were assessed as a loading control (lower panel). ( D ) Representative scheme of the subcellular fractionation procedure. The following fractions were isolated: total homogenate (TH), crude mitochondria (CM), mitochondria-associated ER membranes (MAM), ER-attached mitochondria (MER), bulk ER (ER), and cytosol (Cyt). ( E ) Total levels of STIM1 were analyzed in MAM fraction of HEK293 cells. All fractions were analyzed by immunoblotting (5 μg protein/lane). IP3R1/2/3 were used as an example of ER proteins enriched in MAM, ACSL4 and ERLIN2 were used as MAM markers, p38MAPK as a cytosolic marker and TOM20 as a mitochondrial marker. Fractions from STIM1-KO HEK293 cells were evaluated in separated gels (indicated by the dotted line) as negative control. ( F ) STIM1-KO HEK293 cells stably transfected for the inducible expression of STIM1-GFP (or GFP-empty as a control) were treated with 1 μg/ml doxycycline for 22 h. Immunoprecipitation of GFP-tagged proteins was performed from 1 mg WCL and the co-precipitation of PTPIP51 was assessed by immunoblotting. WCL (3 μg) was loaded as a positive control. Blots are representative of 3 biological replicates. Levels of immunoprecipitated GFP were analyzed as a loading control. ( G ) Total levels of PTPIP51 in WCL were evaluated by immunoblotting (30 μg protein/lane). ( H ) HEK293 cells cultured on collagen-coated coverslips were methanol-fixed and incubated with the specified primary antibodies (rabbit anti-PTPIP51 and/or sheep anti-STIM1) along with the DNA probes rabbit-PLUS and sheep-MINUS. As negative controls, cells were incubated with single primary antibodies. PLA signal (red dots) was analyzed under fluorescence microscopy. The panel shows representative images for all conditions. Scale bar = 10 μm. The graph shows the quantification of interactions (number of red dots) detected by PLA, with the number of cells evaluated in parentheses and the mean of the data represented by the red line. Statistical analysis with unpaired t-test, p < 0.0001. ( I ) Experimental design scheme for fluorescence reconstitution using ddFP. This diagram was created with BioRender.com. ( J ) STIM1-KO HEK293 cells engineered for the expression of inducible STIM1-ddFP-B were transfected for the transient expression of Mito-GA. Reconstitution of the GA–B heterodimer green fluorescence was monitored together with the immunolocalization of TOM20 (AlexaFluor-594). ( K ) Ratio of green (STIM1-mitochondria contacts) and red (mitochondria) fluorescence from 55 cells and 3 independent experiments. Statistical analysis with unpaired t-test, p = 0.0071. .

Article Snippet: Clarified lysates were incubated with 5 μl anti-STIM1 antibody (Cell Signaling Technology, #5668) in binding buffer (10 mM NaH 2 PO 4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100) overnight at 4 °C with gentle rotation.

Techniques: Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot, Positive Control, Control, Incubation, Negative Control, Fractionation, Isolation, Marker, Stable Transfection, Transfection, Cell Culture, Fluorescence, Microscopy

Journal: The EMBO Journal

Article Title: STIM1-containing contact sites promote direct calcium flux from the endoplasmic reticulum to mitochondria

doi: 10.1038/s44318-026-00700-8

Figure Lengend Snippet: ( A ) Quantification of the level of STIM1 in MAM compared with the level in the ER. Data from 5 different subcellular fractionations. Figure shows the result of a representative fractionation. Data are plotted as mean ± S.E.M. ( B , C ) The expression of PTPIP51 and VAPB proteins was assessed in WCL from wild-type U2OS, STIM1-KO U2OS, and STIM1-KO U2OS cells stably expressing STIM1-mCherry (KO + rescue) by immunoblot (30 μg protein/lane). STIM2-KO U2OS and ORAI1-KO U2OS cell lines were also analyzed on a separate gel. In all cases, GAPDH was used as a loading control. ( D , E ) Data from at least 2 biological replicates ( n ≥ 5 technical replicates per cell line) were quantified and plotted as the mean ± S.D. Data were normalized to WT levels. Statistical analysis with unpaired t-test, * p = 0.0135 and ** p = 0.0043. ( F ) Total levels of STIM2 were evaluated by immunoblotting in wild-type and STIM2-KO U2OS cells (30 μg protein/lane). Levels of GAPDH were assessed as a loading control. ( G ) Fura-2-loaded cells were treated with 1 μM Tg in Ca 2+ -free HBSS (added at min = 2). After a 7-min incubation, 2 mM CaCl 2 was added to assess the extent of SOCE. The graph shows the mean F340/F380 ratio ± S.D. over time from 3 independent experiments for WT U2OS (black line, n = 38 cells) and STIM2-KO U2OS cells (red line, n = 50 cells). Statistical analysis with unpaired t-test, p < 0.0001. .

Article Snippet: Clarified lysates were incubated with 5 μl anti-STIM1 antibody (Cell Signaling Technology, #5668) in binding buffer (10 mM NaH 2 PO 4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100) overnight at 4 °C with gentle rotation.

Techniques: Fractionation, Expressing, Stable Transfection, Western Blot, Control, Incubation

Journal: The EMBO Journal

Article Title: STIM1-containing contact sites promote direct calcium flux from the endoplasmic reticulum to mitochondria

doi: 10.1038/s44318-026-00700-8

Figure Lengend Snippet: ( A ) The number of ER-mitochondria contacts was assessed by analyzing the interactions between VAPB (ER) and PTPIP51 (mitochondria) in HEK293 and U2OS cells. Wild-type U2OS, STIM1-KO U2OS, and STIM1-KO U2OS cells stably expressing STIM1-mCherry (KO + rescue) (upper micrographs) were cultured on collagen-coated coverslips and fixed with methanol. Cells were then incubated with the indicated primary antibodies (mouse anti-VAPB and/or rabbit anti-PTPIP51), as well as with the DNA probes rabbit-PLUS and mouse-MINUS. The same assay was performed using HEK293 cells (bottom micrographs), where the rescue condition was represented by the STIM1-KO HEK293 cell line stably transfected for inducible expression of STIM1-GFP. As negative controls, cells were incubated with single primary antibodies. The panel shows representative images for all conditions described, except for negative controls. Scale bar = 10 μm. ( B , C ) Quantification of the interactions (number of red dots) between VAPB and PTPIP51 detected by PLA in U2OS ( B ) and HEK293 ( C ) cells. For the U2OS cell line, STIM2-KO cells were also evaluated. The number of cells evaluated is given in parentheses, and the mean of the data represented by the black line. Statistical analysis with unpaired t-test in all cases. P values are **** p < 0.0001 and p = 0.1027 (n.s.). ( D ) WT or STIM1-KO U2OS cells were transfected for the transient expression of Mito-GA and ER-B. GA–B heterodimer green fluorescence was monitored together with the immunolocalization of TOM20 (AlexaFluor-594). Scale bar = 10 μm. ( E ) Ratio of green (ER-contacts) and red (mitochondria) fluorescence from 27 WT cells or 23 KO cells, and two independent experiments. Statistical analysis with unpaired t-test, p = 0.0071. .

Article Snippet: Clarified lysates were incubated with 5 μl anti-STIM1 antibody (Cell Signaling Technology, #5668) in binding buffer (10 mM NaH 2 PO 4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100) overnight at 4 °C with gentle rotation.

Techniques: Stable Transfection, Expressing, Cell Culture, Incubation, Transfection, Fluorescence

Journal: The EMBO Journal

Article Title: STIM1-containing contact sites promote direct calcium flux from the endoplasmic reticulum to mitochondria

doi: 10.1038/s44318-026-00700-8

Figure Lengend Snippet: ( A ) HEK293 cells were transfected to express the Ca 2+ sensor ER-GCaMP6-210. Scale bar = 10 μm. ( B ) Resting [Ca 2+ ] ER values were defined as the steady-state fluorescence value after the medium conditioning step and before stimulation with 100 μM ATP + 100 μM CCh. Statistical analysis with unpaired t-test, p < 0.0001. ( C ) Values of [Ca 2+ ] ER after Ca 2+ release stimulated by ATP+CCh. Statistical analysis with unpaired t-test, p < 0.0001. ( D ) Decrease in [Ca 2+ ] ER after ATP+CCh stimulus. This decrease was calculated by subtracting the [Ca 2+ ] ER after stimulation with ATP+CCh from the resting [Ca 2+ ] ER . Statistical analysis with unpaired t-test and p = 0.0471. In ( B – D ), the [Ca 2+ ] ER values were monitored in different ROIs from 4 independent experiments. Total number of ROIs analyzed: n = 62 WT and n = 50 KO ROIs. The black line represents the mean of the data. ( E ) Measurement of [Ca 2+ ] i after Ca 2+ release from the ER in WT and STIM1-KO cells. Representative fluorescence profile of fura-2-loaded cells after stimulation with 100 μM ATP + 100 μM CCh in Ca 2+ -free Hank’s balanced salt solution (min = 1). The graph shows the mean F340/F380 ratio ± S.D. over time from 3 independent experiments for WT HEK293 (black line) and 4 experiments for STIM1-KO cells (red line). Total number of cells analyzed: n = 65 WT and n = 89 KO cells. ( F ) Analysis of the maximum increase in the F340/F380 ratio after ATP+CCh addition in Ca 2+ -free assay medium. The maximum values of the F340/F380 ratio of each cell were normalized relative to the resting ratio value to calculate the magnitude of the increase in [Ca 2+ ] i after ER Ca 2+ release. The mean of the data is represented by the black line. Statistical analysis with unpaired t-test. ( G ) Representative fluorescence image and ( H ) fluorescence profile of mito 4× -GCaMP6f after ATP+CCh stimulus. Cells stably transfected for the expression of the mitochondrial Ca 2+ sensor mito 4 × -GCaMP6f were cultured on collagen-coated coverslips and treated with 1 μg/ml doxycycline to induce expression of the Ca 2+ sensor. ER Ca 2+ release was triggered with ATP+CCh in Ca 2+ -free medium, as in ( E ) and ( F ). The ratios of fluorescence at each time point relative to resting fluorescence (F/F r ) were calculated in different ROIs. The graph shows the mean F/F r ± S.E.M. from 4 independent experiments for WT HEK293 ( n = 39 WT ROIs, black line) and STIM1-KO cells ( n = 40 KO ROIs, red line). ( I ) Analysis of the maximum fluorescence increase (F max /F r ) after ATP+CCh. The F max /F r of all ROIs was analyzed from 15 independent experiments for WT cells and 17 experiments for KO cells ( n = 160 WT and n = 169 KO ROIs). The mean of the data is represented by the black line. Statistical analysis with unpaired t-test, p < 0.0001. ( J ) Cells were transiently transfected to express the 4mtD3cpv sensor to measure basal [Ca 2+ ] mit . Data from n = 19 WT cells and n = 16 STIM1-KO cells from 4 independent experiments. The black line represents the mean of data. Statistical analysis with unpaired t-test, p < 0.0001. .

Article Snippet: Clarified lysates were incubated with 5 μl anti-STIM1 antibody (Cell Signaling Technology, #5668) in binding buffer (10 mM NaH 2 PO 4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100) overnight at 4 °C with gentle rotation.

Techniques: Transfection, Fluorescence, Stable Transfection, Expressing, Cell Culture

Journal: The EMBO Journal

Article Title: STIM1-containing contact sites promote direct calcium flux from the endoplasmic reticulum to mitochondria

doi: 10.1038/s44318-026-00700-8

Figure Lengend Snippet: ( A ) Expression of IP3R1, IP3R2, IP3R3, GRP75, VDAC1, and MCU was assessed in WCL from WT HEK293 and STIM1-KO HEK293 cells by immunoblot (40 μg/protein/lane). GAPDH was used as a loading control. ( B – G ) Data from blots were quantified, normalized relative to WT levels, and plotted as the mean ± S.D. from a minimum of 4 technical replicates and at least 2 biological replicates. Statistical analysis with unpaired t-test, p = 0.503 ( B ), p = 0.475 ( C ), p = 0.7186 ( D ), p = 0.1258 ( E ), p = 0.5142 ( F ), p = 0.2384 ( G ). ( H ) Mitochondrial DNA from HEK293 cells was purified and the levels of the mitochondrial gene ND2 were quantified by real-time PCR. Data show the absolute Ct values from 3 independent experiments. Data are plotted as mean ± S.E.M. Statistical analysis with unpaired t-test, p = 0.6488. ( I ) Mitochondrial mass was quantified from HEK293 cells labeled with MitoTracker Green FM and Mitotracker Red FM and analyzed by flow cytometry. Fluorescence signal from 5 independent experiments was normalized to WT values. Data are plotted as mean ± S.E.M. Statistical analysis with unpaired t-test, p = 0.1575 (MitoTracker Green) and p = 0.1611 (MitoTracker Red). .

Article Snippet: Clarified lysates were incubated with 5 μl anti-STIM1 antibody (Cell Signaling Technology, #5668) in binding buffer (10 mM NaH 2 PO 4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100) overnight at 4 °C with gentle rotation.

Techniques: Expressing, Western Blot, Control, Purification, Real-time Polymerase Chain Reaction, Labeling, Flow Cytometry, Fluorescence

Journal: The EMBO Journal

Article Title: STIM1-containing contact sites promote direct calcium flux from the endoplasmic reticulum to mitochondria

doi: 10.1038/s44318-026-00700-8

Figure Lengend Snippet: ( A ) Wild-type HEK293 and STIM1-KO cells were subjected to MAM fractionation, and TH, CM, MAM, and ER fractions were analyzed by immunoblotting (5 μg protein/lane). ACSL4 was used as a loading control for MAM fraction. ( B – D ) Enrichment of IP3Rs in the MAM fraction was quantified relative to total ER levels (MAM + ER signals). Data were normalized to the WT condition and plotted as mean ± S.D. from n = 5 biological replicates for IP3R3 and IP3R2, and n = 3 biological replicates for IP3R1/2/3. Statistical analysis with unpaired t-test in all cases. ( E ) GRP75 and VDAC1 proteins levels were analyzed in the MAM fractions from WT HEK293 and STIM1-KO HEK293 cells. ACSL4 was used as a loading control. ( F , G ) Data were normalized to the WT condition and plotted as mean ± S.D. from n = 4 biological replicates. Statistical analysis with unpaired t-test, p = 0.0288 in ( F ), p = 0.0452 in ( G ). ( H ) Methanol-fixed HEK293 cells were incubated with the indicated primary antibodies (rabbit anti-VDAC1 and/or mouse anti-IP3R1/2/3) as well as the DNA probes rabbit-PLUS and mouse-MINUS. As negative controls, cells were incubated with individual primary antibodies. Representative images for all the conditions are shown. Scale bar = 10 μm. ( I ) Quantification of the assay from ( H ), with the number of cells analyzed indicated in parentheses. The black line represents the mean of the data. Statistical analysis with unpaired t-test, **** p < 0.0001, * p = 0.0139. .

Article Snippet: Clarified lysates were incubated with 5 μl anti-STIM1 antibody (Cell Signaling Technology, #5668) in binding buffer (10 mM NaH 2 PO 4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100) overnight at 4 °C with gentle rotation.

Techniques: Fractionation, Western Blot, Control, Incubation

Journal: The EMBO Journal

Article Title: STIM1-containing contact sites promote direct calcium flux from the endoplasmic reticulum to mitochondria

doi: 10.1038/s44318-026-00700-8

Figure Lengend Snippet: ( A ) Representative whole-cell Seahorse plot of the oxygen consumption rate (OCR, pmol O 2 /min/cell). Wild-type and STIM1-KO U2OS cells were cultured on a Seahorse microplate for 24 h (20,000 cells/well). OCR was monitored under basal conditions and after the sequential addition of oligomycin, FCCP, and antimycin A + rotenone (A + R). After the assay, cells were counted in each well, and OCR values were normalized accordingly. Data are presented as mean ± S.D. from a single experiment, with n = 3 WT (black line) and n = 5 KO (red line) technical replicates. ( B – D ) Quantification of mitochondrial parameters derived from Seahorse assay analysis. The average values from at least 3 technical replicates across 4 independent experiments were evaluated. Statistical analysis was performed using a paired t-test ratio to assess the differences between pairs. ( B ) Maximal respiration: the maximum rate measurement after FCCP injection minus the non-mitochondrial respiration. * p = 0.0356. Data are plotted as mean ± S.D. ( C ) ATP production: the last rate measurement before oligomycin injection minus the minimum rate measurement after oligomycin injection. * p = 0.0472. Data are plotted as mean ± S.D. ( D ) Spare respiratory capacity: the maximal respiration rate minus the basal respiration. * p = 0.0446. Data are plotted as mean ± S.D. ( E ) Whole cell lysates from WT HEK293 and STIM1-KO HEK293 cells were obtained, and samples containing 20 μg protein were assessed by immunoblotting. Expression levels of proteins from all mitochondrial complexes were analyzed. NDUFB8 (CI), SDHB (CII), UQCRC2 (CIII), and ATP5A (CV) proteins were evaluated using total OXPHOS commercial antibody cocktail, while a specific anti-COX IV antibody was used for CIV assessment. GAPDH expression served as a loading control. ( F – J ) Quantification of the expression levels of all mitochondrial proteins shown in ( E ). The average of n ≥ 2 technical replicates from 3 biological replicates, normalized to WT condition, is plotted in all cases. Statistical analysis with unpaired t-test, **** p < 0.0001. Data are plotted as mean ± S.D. ( F ) NDUFB8 protein (CI). ( G ) SDHB protein (CII). ( H ) UQCRC2 protein (CIII). (I) COX IV protein (CIV). ( J ) ATP5A protein (CV). ( K ) HEK293 cells were cultured on collagen-coated coverslips for 48 h, incubated with 5 μM CellROX™ for 30 min, washed, and maintained in HBSS for 10 min. A minimum of 4 images were acquired per experiment. Three independent experiments were conducted. Fluorescence intensity per cell after background subtraction. The number of cells evaluated is indicated in parentheses, and the mean of the data represented by the black line. Statistical analysis with unpaired t-test, **** p < 0.0001. ( L ) HEK293 (WT and STIM1-KO) cell lysates were assessed for phospho-Ser293 pyruvate dehydrogenase α1 (p-PDH) and total PDH by immunoblotting. As a negative control, cells were treated with 20 mM dichloroacetate (DCA) for 24 h before lysis. ( M ) Quantification of p-PDH levels from 4 independent experiments ( n = 11 total replicates) for non-DCA-treated samples, and 2 independent experiments for DCA-treated samples ( n = 4 total replicates). Statistical analysis with unpaired t-test, *** p = 0.0007 and * p = 0.0165. Data are plotted as mean ± S.E.M. ( N ) HEK293 cells were labeled with MitoTracker Green FM, MitoTracker Red CMXRos, and DAPI for viability assessment. Fluorescence recordings were normalized to the values of WT cells and plotted accordingly. Data from 5 independent experiments are shown as scatter dot plot. Statistical analysis with unpaired t-test, ** p = 0.0019. ( O ) The ratio between the signal from the potential-sensitive MitoTracker Red CMXRos over the signal from potential-insensitive MitoTracker Green is shown as a readout of mitochondrial potential, normalized to mitochondrial mass. Data from 5 independent experiments are shown as scatter dot plot. Statistical analysis with unpaired t-test, * p = 0.0192. .

Article Snippet: Clarified lysates were incubated with 5 μl anti-STIM1 antibody (Cell Signaling Technology, #5668) in binding buffer (10 mM NaH 2 PO 4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100) overnight at 4 °C with gentle rotation.

Techniques: Cell Culture, Derivative Assay, Injection, Western Blot, Expressing, Control, Incubation, Fluorescence, Negative Control, Lysis, Labeling

Journal: The EMBO Journal

Article Title: STIM1-containing contact sites promote direct calcium flux from the endoplasmic reticulum to mitochondria

doi: 10.1038/s44318-026-00700-8

Figure Lengend Snippet: ( A ) STIM1-KO HEK293 cells inducibly expressing STIM1-GFP, or GFP only (empty GFP-vector) as a control, were washed twice in Ca 2+ -free HBSS and incubated with 1 μM Tg in Ca 2+ -free HBSS for 2 min. Cells from the SOCE-free condition (-Ca 2+ ) were then lysed. Cells from the SOCE-allowed condition (+Ca 2+ ) were incubated for an additional 2 min with 1 μM Tg + 2 mM CaCl before lysis. Immunoprecipitation of GFP-tagged proteins was performed using 1 mg WCL, and the co-precipitation of GRP75 was evaluated by immunoblotting. A fraction of WCL from the untreated condition (3 μg) was loaded as a positive control. Levels of immunoprecipitated GFP were assessed as a loading control. Levels of total GRP75 and total STIM1-GFP were evaluated from WCL by immunoblotting (30 μg protein/lane) and are shown in Fig. . ( B ) Quantification of co-precipitated GRP75 (from 4 independent experiments for resting and Tg-treated samples, 2 experiments for SOCE-allowed samples). Statistical analysis with unpaired t-test. Control vs Tg, p = 0.0021; Control vs Tg+Ca 2+ , p = 0.0038. Data are plotted as mean ± S.D. ( C ) Evaluation of ER-mitochondria contacts after Tg treatment by PLA. Wild-type HEK293 cells were washed twice with Ca 2+ -free HBSS and incubated with 1 μM Tg in this medium for 2 min, as in ( A ). Cells were then fixed and incubated with mouse anti-VAPB and rabbit anti-PTPIP51 antibodies, as well as with the DNA probes rabbit-PLUS and mouse-MINUS. The untreated control was fixed with methanol directly from the culture medium, without any washing step. The interaction of proteins (red dots) was analyzed under fluorescence microscopy. Representative images are shown in the figure. Scale bar = 10 μm. Negative controls are shown in Fig. . ( D ) Quantification of the interactions detected by PLA, with the number of cells evaluated in parentheses. The mean of the data is represented by the black line. Statistical analysis with unpaired t-test, **** p < 0.0001. ( E ) Analysis of the STIM1-GRP75 interaction during ER Ca 2+ release triggered by ATP+CCh. Cells were washed twice with HBSS, then twice with Ca 2+ -free HBSS, and incubated with 100 μM ATP + 100 μM CCh in Ca 2+ -free HBSS for 15–30 s before lysis. Immunoprecipitation of GFP-tagged proteins was performed using 1 mg WCL, and co-precipitated GRP75 was analyzed by immunoblotting. For the untreated control (0-s condition), lysis was conducted immediately after the first two washes with HBSS. Levels of immunoprecipitated GFP were analyzed as a loading control. Total levels of GRP75 in WCL were assessed by immunoblotting (30 μg protein/lane) and are shown in Fig. . ( F ) Quantification of co-precipitated GRP75 from 2 independent experiments and 4 technical replicates. Statistical analysis with unpaired t-test, **** p < 0.0001 and p = 0.2266 (n.s.). Data are plotted as mean ± S.D. ( G ) Quantification of ER-mitochondria contacts in U2OS cells expressing Mito-GA and ER-B, as described in Fig. . Reconstitution of green fluorescence was normalized to the total mitochondrial mass, revealed with anti-TOM20 + AlexaFluor 594 (red) immunofluorescence. Data show the mean (black line) from two independent experiments. Statistical analysis with unpaired t-test, p = 0.0014. ( H ) HEK293 cells were washed twice with HBSS, then twice with Ca 2+ -free HBSS, and incubated with 100 μM ATP + 100 μM CCh in Ca 2+ -free HBSS for 30 s before isolation of MER, TH, CM, MAM, and ER. For the untreated control (0-s condition), lysis was conducted immediately after the first two washes with HBSS. STIM1 and GRP75 levels were analyzed by immunoblotting. ACSL4 and ERLIN2 were assessed as positive controls for MAMs, while VDAC1 and TOM20 were assessed as positive controls for MER and CM. In all cases, 7 μg protein was loaded in each lane. ( I ) Top panels: Quantification of GRP75 and STIM1 in MAMs was performed using data from 4 independent subcellular fractionation experiments. For normalization, GRP75 levels in MAMs were normalized to levels in TH, and STIM1 levels in MAMs were normalized to levels in ER. Bottom panels: Quantification of STIM1 and GRP75 levels in TH. Data are normalized to resting conditions. Statistical analysis with unpaired t-test. p < 0.0001 for GRP75 in MAM, p = 0.0003 for STIM1 in MAM, p = 0.0757 for total GRP75, and p = 0.1220 for total STIM1. Data are plotted as mean ± S.E.M. ( J ) Co-immunoprecipitation assays were performed using 1 mg WCL from STIM1-KO HEK293 cells inducibly expressing STIM1-GFP, STIM1(R429C)-GFP, or GFP only (empty GFP vector) as a control. The co-precipitation of GRP75 was evaluated by immunoblotting. A fraction of WCL (3 μg) from cells expressing STIM1-GFP was loaded as a positive control. Blots are representative of 3 biological replicates. Levels of immunoprecipitated GFP were assessed as a loading control. Total levels of GRP75 from WCL were analyzed by immunoblotting (30 μg protein/lane) and are shown in Fig. . ( K ) Quantification of the co-precipitation data ( n = 6 technical replicates from 3 biological replicates) is shown. Data were normalized to immunoprecipitated GFP levels and plotted as mean ± S.D. Statistical analysis with unpaired t-test, p < 0.0001. ( L ) STIM1-KO HEK293 cells inducibly expressing STIM1-GFP or STIM1(R429C)-GFP, labeled as WT or R429C in the graph, were fixed and incubated with mouse anti-VAPB and rabbit anti-PTPIP51 antibodies and the DNA probes rabbit-PLUS and mouse-MINUS. Scale bar = 10 μm. Negative controls are shown in Fig. . ( M ) Quantification of the number of interactions per cell. The number of cells analyzed is indicated in parentheses, and the mean of data is represented by the black line. Statistical analysis with unpaired t-test, p < 0.0001. .

Article Snippet: Clarified lysates were incubated with 5 μl anti-STIM1 antibody (Cell Signaling Technology, #5668) in binding buffer (10 mM NaH 2 PO 4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100) overnight at 4 °C with gentle rotation.

Techniques: Expressing, Plasmid Preparation, Control, Incubation, Lysis, Immunoprecipitation, Western Blot, Positive Control, Fluorescence, Microscopy, Immunofluorescence, Isolation, Fractionation, Labeling

Journal: The EMBO Journal

Article Title: STIM1-containing contact sites promote direct calcium flux from the endoplasmic reticulum to mitochondria

doi: 10.1038/s44318-026-00700-8

Figure Lengend Snippet: ( A ) Data supporting co-immunoprecipitation experiment shown in Fig. : Levels of total GRP75 and total STIM1-GFP were evaluated from WCL by immunoblotting (30 μg protein/lane). ( B ) Data supporting co-immunoprecipitation experiment shown in Fig. : Levels of total GRP75 and total STIM1-GFP were evaluated from WCL by immunoblotting (30 μg protein/lane). ( C ) STIM1-KO HEK293 cells inducibly expressing Flag-STIM1 (black line) or Flag-STIM1(R429C) (red line) were incubated with fura-2-AM for 45 min. The F340/F380 ratio was then recorded in Ca 2+ -free HBSS. After 2 min, cells were treated with 1 μM Tg, and 4 min later, 2 mM CaCl 2 was added to evaluate SOCE. Data are shown as the mean F340/F380 ratio ± S.D. from 2 independent experiments ( n = 24 cells) for each cell line. Statistical analysis with unpaired t-test, p < 0.0001. ( D ) Level of [Ca 2+ ] ER in cells expressing STIM1(R429C). STIM1-KO HEK293 cells stably transfected for the expression of Flag-STIM1 (WT) or Flag-STIM1(R429C) were transiently transfected for the expression of the Ca 2+ sensor ER-GCaMP6-210. The graph shows basal [Ca 2+ ] ER values calculated from 3 independent experiments for cells expressing Flag-STIM1 ( n = 38 WT ROIs) and from 4 experiments for cells expressing Flag-STIM1(R429C) ( n = 53 R429C ROIs). The mean of data is represented by the black line. Statistical analysis with unpaired t-test. ( E ) HEK293 cells stably transfected for the inducible expression of STIM1(WT)-GFP or STIM1(R429C)-GFP were treated with 1 μM Tg in Ca 2+ -free HBSS for 10 min, then fixed and mounted for microscopy analysis of GFP fluorescence. Images are representative of 3 independent experiments. Scale bar = 10 μm. ( F ) Data supporting co-immunoprecipitation experiment shown in Fig. : Total levels of GRP75 and STIM1-GFP from WCL were analyzed by immunoblotting (30 μg protein/lane). .

Article Snippet: Clarified lysates were incubated with 5 μl anti-STIM1 antibody (Cell Signaling Technology, #5668) in binding buffer (10 mM NaH 2 PO 4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100) overnight at 4 °C with gentle rotation.

Techniques: Immunoprecipitation, Western Blot, Expressing, Incubation, Stable Transfection, Transfection, Microscopy, Fluorescence

Journal: The EMBO Journal

Article Title: STIM1-containing contact sites promote direct calcium flux from the endoplasmic reticulum to mitochondria

doi: 10.1038/s44318-026-00700-8

Figure Lengend Snippet: ( A ) Scheme of full-length STIM1 and STIM1 with C-terminal deletions. The following domains of STIM1 are shown: Ca 2+ -sensitive EF-hand domain (cEF), sterile α motif (SAM), transmembrane domain (TM), coiled-coil domains (CC1-3), C-terminal inhibitory domain (CTID), and lysine-rich domain (K). The figure shows the deleted sequences and C-terminal domains in each mutant (named on the left), as reported elsewhere (Sanchez-Lopez et al, ). This scheme was created with BioRender.com. ( B ) Pull-down assays of GFP-tagged proteins were performed using 1 mg WCL from STIM1-KO HEK293 inducibly expressing full-length STIM1-GFP, STIM1(Δ235-442)-GFP (mutant 1), STIM1(Δ443-550)-GFP (mutant 2), STIM1(Δ551-685)-GFP (mutant 3), or GFP, as a control. Co-precipitation of GRP75 was assessed by immunoblot. A fraction of WCL (3 μg) from cells expressing STIM1-GFP was loaded as a positive control. Blots are representative of 3 biological replicates. Total pulled-down GFP was used as a loading control. ( C ) Total levels of STIM1 (either mutant or WT) and total levels of GRP75 in WCL were analyzed by immunoblotting (30 μg protein/lane). ( D , E ) Data supporting co-immunoprecipitation experiment shown in Fig. B and , respectively: Total levels of GRP75 and STIM1-GFP from WCL were analyzed by immunoblotting (30 μg protein/lane). ( F ) HEK293 cells stably transfected for the inducible expression of STIM1(WT)-GFP or STIM1(Δ551-611)-GFP were treated with 1 μM Tg in Ca 2+ -free HBSS for 10 min, then fixed and mounted for microscopy analysis of GFP fluorescence. Images are representative of 3 independent experiments. Scale bar = 10 μm. ( G ) STIM1-KO HEK293 cells inducibly expressing either Flag-STIM1(full-length) or Flag-STIM1(Δ551-611) were incubated with fura-2-AM. The F340/F380 ratio was then recorded in Ca 2+ -free HBSS. After 2 min, cells were treated with 1 μM Tg, and after a 5-min incubation, 2 mM CaCl 2 was added to the assay medium to evaluate SOCE. Data represent the mean ± S.D. of the F340/F380 over time from 3 independent experiments for cells expressing Flag-STIM1 (black line, n = 33 cells) and Flag-STIM1(Δ551-611) (red line, n = 35 cells). ( H ) Immunoprecipitation assay (1 mg WCL) from STIM1-KO HEK293 cells inducibly expressing STIM1-GFP (labeled as FL), the STIM1(551-611)-GFP peptide, or GFP as a control. GRP75 co-precipitation was analyzed by immunoblotting. Levels of immunoprecipitated GFP were evaluated as a loading control. ( I ) Total levels of GRP75 and GFP-tagged proteins in the WCL used for the co-immunoprecipitation shown in ( H ) were assessed by immunoblotting (30 μg protein/lane). ( J ) Supporting data for the co-immunoprecipitation experiment shown in Fig. : total levels of GRP75 and STIM1-GFP in WCL were analyzed by immunoblotting (30 μg protein/lane). ( K ) HEK293 cells stably transfected for the inducible expression of Flag-STIM1(551-611) (referred to as peptide), or Flag-empty vector as a control, were transiently transfected for the expression of the Ca 2+ sensor ER-GCaMP6-210. The graph shows basal (steady-state) [Ca 2+ ] ER in both cell lines. The mean of data is represented by the black line. Statistical analysis with unpaired t-test. ( L ) [Ca 2+ ] ER values after Ca 2+ release stimulated with ATP+CCh. Statistical analysis with unpaired t-test. ( M ) Decrease in [Ca 2+ ] ER following ATP+CCh stimulus. In ( K – M ), [Ca 2+ ] ER values were calculated from 3 independent experiments for cells expressing Flag-(empty tag) ( n = 36 ROIs) and from 4 experiments for cells expressing Flag-peptide 551-611 ( n = 47 ROIs). Statistical analysis with unpaired t-test. .

Article Snippet: Clarified lysates were incubated with 5 μl anti-STIM1 antibody (Cell Signaling Technology, #5668) in binding buffer (10 mM NaH 2 PO 4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100) overnight at 4 °C with gentle rotation.

Techniques: Sterility, Mutagenesis, Expressing, Control, Western Blot, Positive Control, Immunoprecipitation, Stable Transfection, Transfection, Microscopy, Fluorescence, Incubation, Labeling, Plasmid Preparation

Journal: The EMBO Journal

Article Title: STIM1-containing contact sites promote direct calcium flux from the endoplasmic reticulum to mitochondria

doi: 10.1038/s44318-026-00700-8

Figure Lengend Snippet: ( A ) Schematic representation of STIM1 mutants with deletions in the 551-685 region. The specific sequences deleted between the amino acids 551 and 685 for each of the generated STIM1 mutants are shown. This diagram was created with BioRender.com. ( B ) Immunoprecipitation of GFP-tagged proteins was performed using 1 mg WCL from STIM1-KO HEK293 inducibly expressing STIM1-GFP, STIM1(Δ551-685)-GFP (mutant 3), STIM1(Δ551-642)-GFP (mutant 4), STIM1(Δ643-677)-GFP (mutant 7), STIM1(Δ668-674)-GFP (mutant 8), STIM1(Δ672-685)-GFP (mutant 9), or GFP (empty vector) as a control. Co-precipitation of GRP75 was assessed by immunoblotting. WCL (3 µg) from cells expressing STIM1-GFP was loaded as a positive control. Total immunoprecipitated GFP was used as a loading control. Total levels of STIM1 and GRP75 in WCL (30 μg protein/lane) were analyzed by immunoblotting (Fig. ). ( C ) Immunoprecipitation of GFP-tagged proteins was performed using 1 mg WCL from STIM1-KO HEK293 inducibly expressing STIM1-GFP, STIM1(Δ551-685)-GFP (mutant 3), STIM1(Δ551-642)-GFP (mutant 4), STIM1(Δ551-611)-GFP (mutant 5), STIM1(Δ582-642)-GFP (mutant 6), STIM1(Δ672-685)-GFP (mutant 9), or GFP only as a control. Other conditions as stated for ( B ). Total levels of STIM1 and GRP75 in WCL were analyzed by immunoblotting (30 μg protein/lane) (Fig. ). ( D ) STIM1-KO HEK293 cells inducibly expressing full-length (FL) STIM1-GFP or STIM1(Δ551-611)-GFP were fixed and incubated with rabbit anti-GRP75 and/or sheep anti-GFP antibodies. Negative controls consisted of cells incubated with single primary antibodies. Scale bar = 10 μm. ( E ) Quantification of the interactions per cell detected by PLA in ( D ). The number of cells evaluated is indicated in parentheses, and the mean is represented by the black line. Statistical analysis with unpaired t-test, p < 0.0001 and p = 0.5354 (n.s.). ( F ) STIM1-KO HEK293 cells expressing STIM1-GFP (full-length, FL) or STIM1(Δ551-611)-GFP were incubated with a rabbit anti-PTPIP51 and mouse anti-VAPB antibody for a PLA assay. Negative controls are shown in Fig. . Scale bar = 10 μm. ( G ) Quantification of the PLA assay (number of red dots per cell) shown in ( F ). The number of cells evaluated is indicated in parentheses, and the black line represents the mean of the data. Statistical analysis with unpaired t-test, p = 0.0016. ( H ) HEK293 cells expressing STIM1(full-length)-GFP were transfected for transient expression of a Flag-tagged STIM1 peptide encompassing residues 551-611. After 24 h of transfection, STIM1-GFP was immunoprecipitated and co-precipitated GRP75 was assessed by immunoblotting. Total levels of GRP75 and STIM1-GFP from WCL are shown in Fig. . ( I ) Quantification of co-precipitated GRP75 from 2 independent experiments and 3 technical replicates. Statistical analysis with unpaired t-test, p = 0.0072. Data are plotted as mean ± S.D. ( J ) HEK293 cells stably and inducibly expressing mito 4× -GCaMP6f were transiently transfected for the expression of mCherry-tagged STIM1 peptide corresponding to residues 551-611. The peptide was tagged with mCherry to select transfected cells, and the ratios F/Fr were calculated in mCherry-expressing cells only. The graph shows Fmax/Fr after the addition of ATP+CCh in Ca 2+ -free medium. Statistical analysis with unpaired t-test, p = 0.0407. Dots represent data from individual cells. ( K ) Analysis of the area under the curve (AUC) of the fluorescence profile after the ATP+CCh stimulus. A total of 16 independent experiments were performed in control (WT) cells ( n = 67 cells) and 17 experiments for cells expressing the peptide (551-611)-mCherry ( n = 44 cells). The mean of the data is represented by the black line. Statistical analysis with unpaired t-test, p = 0.0021. ( L ) HEK293 cells inducibly expressing the Flag-tagged STIM1(551-611)-peptide or HEK293 control cells (with no peptide expression) were fixed and incubated with anti-VAPB and anti-PTPIP51 antibodies and PLA reagents, as in ( F ). Scale bar = 10 μm. ( M ) Quantification of VAPB-PTPIP51 interactions per cell from ( L ). The number of cells evaluated is indicated in parentheses, and the black line represents the mean of the data. Statistical analysis with unpaired t-test, p < 0.0001. ( N ) Wild-type HEK293 cells inducibly expressing the Flag-STIM1(551-611) peptide (red line) and loaded with fura-2 were assessed for the extension of SOCE and compared to cells with no peptide expression (black line). A total number of 51 (WT) and 49 (WT + peptide) cells were analyzed from 3 independent experiments. Data are plotted as mean ± S.D. .

Article Snippet: Clarified lysates were incubated with 5 μl anti-STIM1 antibody (Cell Signaling Technology, #5668) in binding buffer (10 mM NaH 2 PO 4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100) overnight at 4 °C with gentle rotation.

Techniques: Generated, Immunoprecipitation, Expressing, Mutagenesis, Plasmid Preparation, Control, Western Blot, Positive Control, Incubation, Transfection, Stable Transfection, Fluorescence

Journal: The EMBO Journal

Article Title: STIM1-containing contact sites promote direct calcium flux from the endoplasmic reticulum to mitochondria

doi: 10.1038/s44318-026-00700-8

Figure Lengend Snippet: In this model, GRP75 recruitment requires STIM1 in its closed conformation, reflecting an ER fully replete with intraluminal Ca 2+ . Under these conditions, GRP75 couples to IP3Rs and VDAC to promote Ca 2+ transfer. This efflux, however, induces a localized and partial ER Ca 2+ depletion, triggering a conformational change in STIM1 and the dissociation of GRP75 from the IP3R–VDAC complex and MAMs, thereby terminating Ca 2+ transfer and preventing mitochondrial Ca 2+ overload.

Article Snippet: Clarified lysates were incubated with 5 μl anti-STIM1 antibody (Cell Signaling Technology, #5668) in binding buffer (10 mM NaH 2 PO 4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100) overnight at 4 °C with gentle rotation.

Techniques: