tom20 polyclonal  (Proteintech)

Journal: bioRxiv

Article Title: Mitochondria – insulin granule crosstalk controls the early stages of granule maturation

doi: 10.64898/2026.02.23.707428

Figure Lengend Snippet: Interactions between newly synthesized granules and mitochondria (A) Confocal microscopy image of a MIN6 cell expressing NPY-mNeonGreen (cyan) and Tom20-mApple (yellow). Pixel overlaps between cyan and yellow is shown in magenta.(B) Fraction of NPY-mNeonGreen-containing granules in close proximity to mitochondria (Tom20-mApple) (n=12 cells from three experiments; means ± SEM; Student’s paired t-test; ****P<0.0001).(C) Montage of images from a live cell showing the spatio-temporal relationship between granules (cyan) and mitochondria (yellow). Pictures in magenta show pixel overlap between cyan and yellow. (D) Workflow for pulse-chase labeling of insulin granules based on age (top) and confocal microscopy images of a MIN6 cells expressing NPY-Halo and labeled according to the workflow followed by immunostaining against the mitochondrial marker Tom20 (cyan). Scale bars: 5 μm. (E) Mander’s coefficients denoting the fraction of Tom20 overlapping with new (JFX549- labeled) and old (JFX650-labeled) insulin granules (means ± SEM, ****P<0.001; Kruskal–Wallis test; n = 30 cells). (F) Cartoon showing the principle of the RUSH system. (G) Confocal microscopy images of MIN6 cells expressing spGFP-proCpepRUSH (green) and the mitochondrial marker Tom20-mApple (magenta). Images are from 0, 30, 60 and 120 min post-biotin addition (200 μM). (H) Relative mitochondria enrichment on spGFP-proCpep-positive granules 60 and 120 min after biotin addition (n=111 cells; three experiments; means ± SEM; Student’s unpaired t-test; ** P<0.01). (I) Cartoon showing the principle behind detection of granule-mitochondria contacts in live cells using the split-fluorophore RA-GB system. (J) Confocal microscopy images of a MIN6 cell expressing the granule marker NPY- Halo JFX650 and the granule-mitochondria proximity detector (magenta). Boxed area is magnified to the left and shows how mitochondria-granule proximities are restricted to a small surface on the granule. (K) Fraction of granules in proximity to mitochondria based on their subcellular localization (peripheral or central) (n=8 cells; three experiments; means ± SEM; Student’s paired t-test; **P<0.01). (L) FIB-SEM images showing the proximity between insulin granules and mitochondria in a mouse β-cell. Scale bar: 200 nm. (M) Confocal microscopy images of mouse islets with insulin immunostaining shown in green and mitochondria-granule proximity sites detected with PLA, using antibodies against the mitochondria (VDAC) and insulin granules (Rab3a), shown in red. In control experiments, only the VDAC antibody was used. Scale bar: 50 μm. (N) Average number of PLA puncta per insulin-positive cell (single confocal plane; means ± SEM, n = 14, >30 islets for each experiment, unpaired, two-tailed Student’s t test, **p < 0.01).

Article Snippet: The following antibodies and dyes were used in the study: Rab3 monoclonal (catalog no. 107 111, Synaptic Systems, host: mouse, 1:500), TOM20 polyclonal (catalog no. 11802-1-AP, Proteintech, host: Rabbit, 1:400), insulin polyclonal (catalog no. A0564, Dako, host: guinea pig, 1:1000), VDAC (catalog no. MA533205, host: rabbit, 1:200, Invitrogen), VDAC1 (catalog no. 66345-1-lg, Proteintech, host: mouse, 1:200), VDAC2 (catalog no. 66388-1-lg, Proteintech, host: mouse, 1:200), VNUT polyclonal (catalog no. ABN83, Merck, host: guinea pig, 1:200).

Techniques: Synthesized, Confocal Microscopy, Expressing, Pulse Chase, Labeling, Immunostaining, Marker, Control, Two Tailed Test

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: After 24 h, cells were fixed with 4% paraformaldehyde and subjected to immunolocalization using a rabbit polyclonal anti-TOM20 antibody (Proteintech #11802-1-AP) followed by an AlexaFluor 594-conjugated secondary antibody.

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 ) 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: After 24 h, cells were fixed with 4% paraformaldehyde and subjected to immunolocalization using a rabbit polyclonal anti-TOM20 antibody (Proteintech #11802-1-AP) followed by an AlexaFluor 594-conjugated secondary antibody.

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 ) 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: After 24 h, cells were fixed with 4% paraformaldehyde and subjected to immunolocalization using a rabbit polyclonal anti-TOM20 antibody (Proteintech #11802-1-AP) followed by an AlexaFluor 594-conjugated secondary antibody.

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

Journal: iScience

Article Title: HMOX1 drives dihydroartemisinin-sensitized ferroptosis antagonized by mitochondrial fusion

doi: 10.1016/j.isci.2025.114382

Figure Lengend Snippet: Mitochondrial oxidative stress induced by DHA and RSL-3 activates mitochondrial fusion (A) The thumbnail sketch of mitochondrial functions that may be regulated by mitochondrial oxidation. (B) Cell viability of N27 cells treated with DHA (1.5 μM) and RSL-3 (100 nM) for 48 h in the absence or presence of mitochondrial regulators (2 μM oligo A, 2 μM CCCP, 10 μM αKG, 1 μM rotenone), n = 6 wells from one representative of two independent experiments. (C–E) Western blot and quantifications of the OXPHOS, Tom20, β-actin, and GAPDH expression in N27 cells treated with DHA (1.5 μM) and RSL-3 (100 nM) for 12 h. (F–I) Western blot and quantifications of the MFN1, MFN2, DRP1, and GAPDH expression in N27 cells treated with DHA (1.5 μM) and RSL-3 (100 nM) for 12 h. (J) N27 cells were treated with DHA (1.5 μM) and RSL-3 (100 nM) for 12 h and detected by JC-1 using flow cytometry. Statistical analysis of the ratio of the MFI of JC-1 red to JC-1 green is shown, n = 6 wells from one representative of two independent experiments. (K–M) Western blot and quantifications of the MFN1, MFN2, and GAPDH expression in N27 cells treated with MitoQ (5 μM), DHA (1.5 μM), and RSL-3 (100 nM) for 12 h. (N) Cell viability of N27 cells treated with DHA (1.5 μM) and RSL-3 (100 nM) in the absence or presence of mitochondrial fusion promoter M1 (5 μM) for 48 h, n = 6 wells from one representative of two independent experiments. Data are means ± SEM, n = 3 wells from one representative of two independent experiments unless specified. One-way ANOVA was performed unless specified.

Article Snippet: TOM20 Polyclonal antibody (1:5000) , Proteintech , 11802-1-AP; RRID: AB_2207530.

Techniques: Western Blot, Expressing, Flow Cytometry

Journal: Nature Communications

Article Title: Automatic optimization of flat-field corrections by evaluation and enhancement (EVEN) in multimodal optical microscopy

doi: 10.1038/s41467-025-68150-0

Figure Lengend Snippet: A three-channel fluorescence microscopy measurement of stained HEK293 cells measured by Ph2 objective is automatically optimized by EVEN (prediction dataset 2, red: peroxisomal proteins (anti-GFP nanobody); green: TOMM20 protein; blue: peroxisomal proteins (eGFP)). a Raw multi-channel image. The inset shows the 2 × 2 tile section of the image used in this figure, with dashed white lines marking tile borders. Multiple corrections are obtained by applying BaSiC, CIDRE, Fourier methods, and then optimizing the multi-channel image with EVEN. EVEN selects CIDRE for the red and green channel, and Fourier for the blue channel. b Steps to analyse the measurements of stained cells: multi-channel images are converted to greyscale by summing the single channels (that contain signals from different components of the cytoplasm) and are analysed with automatic cells segmentation using Cellpose . The greyscale image is obtained for the raw measurement, the single-channel corrections and the EVEN optimization. c Intensity sum (along y) of the greyscale inset for each method. The black dashed line indicates the border between neighbouring tiles. The corrected images show higher intensities at the edges of the tiles and the enhancement of sample features. EVEN and CIDRE show the greatest intensity recovery between tiles. d Top row: multi-channel images obtained with single-method corrections and EVEN optimization; the white dashed boxes highlight two regions significantly improved by EVEN. Bottom row: Cellpose prediction on the greyscale sum of the three channels for each method. After correction of uneven illumination, Cellpose can outline a greater number of cells, especially at the borders of neighbouring tiles. White dashed boxes highlight three regions where EVEN optimization provides better identification of the cells compared to non-optimized images. Bottom labels show, for each image, the normalized EVEN score summed over three channels and the cell count in the zoomed region. While counts are not strictly correlated with segmentation performance, good correction of uneven illumination enhances downstream analysis and generally increases the number of detected cells. Further quantification is provided in Supplementary Fig. . Scale bar: 180 µm, size of a single tile.

Article Snippet: Additionally, immunolabeling was performed on TOMM20-protein with anti-Tomm20 rabbit polyclonal antibodies (proteintech, USA), dilution 1:200, and goat anti-rabbit IgG secondary antibodies labelled with Abberior STAR Orange (Abberior, Germany) at a dilution of 1:350.

Techniques: Fluorescence, Microscopy, Staining, Cell Counting

Journal: Molecular Medicine

Article Title: The SARS-CoV-2 nucleocapsid protein induces microglia senescence-mediated cognitive impairment via Glycolysis

doi: 10.1186/s10020-025-01410-3

Figure Lengend Snippet: SARS-CoV-2 N protein drives the senescence of BV2 microglial cells by triggering mitochondrial dysfunction. BV2 microglial cells were treated with Mdivi-1 (100 nM) 30 min before Codon-optimized pLJM1-SARS-CoV-2 N-FLAG transfection. A Representative image of BV2 cells loaded with the mitochondrial membrane potential indicator JC-1 (bar = 20 μm). B Mitochondrial morphology stained with TOM20 in BV2 cells (bar = 2 μm). C - D The expression levels of p-DRP1 and DRP1 proteins in BV2 microglial cells detected by Western blot ( n = 3). Bar graph data represent the mean ± SD of the target protein intensity (normalized to DRP1) in the experimental group relative to that in the control group. E Representative images of BV2 cells loaded with the mitochondrial membrane potential indicator JC-1 (bar = 20 μm). F The expression levels of p19 , p21 , p53 , Il-1β , and Tnf-α mRNA in BV2 microglial cells detected by real-time PCR ( n = 3). G SA-β-gal staining was performed after transfection of Codon-optimized pLJM1-SARS-CoV-2 N-FLAG and treatment with Mdivi-1 for 36 h (bar = 10 μm). H The fluorescence intensity of Ki67 (green) was detected by immunofluorescence (bar = 50 μm). I - J The expression levels of p-DRP1, p53, p21, p16, and γ-H2AX proteins in BV2 microglial cells were detected by Western blot ( n = 3). Bar graph data represent the mean ± SD of the target protein intensity (normalized to α-tubulin) in the experimental group relative to that in the control group. Comparisons between the two groups were made with an unpaired t -test. Differences among multiple groups were performed using ANOVA. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Rabbit anti-TOM20 polyclonal antibody , Proteintech , 11802-1-AP , 1: 200.

Techniques: Transfection, Membrane, Staining, Expressing, Western Blot, Control, Real-time Polymerase Chain Reaction, Fluorescence, Immunofluorescence