mouse anti vdac1  (Proteintech)

Journal: iScience

Article Title: Spastin regulates ER-mitochondrial contact sites and mitochondrial homeostasis

doi: 10.1016/j.isci.2024.110683

Figure Lengend Snippet: Spastin partially colocalizes with mitochondria and accumulates in MERCs (A) Representative images of HeLa cells cotransfected with M1-GFP (green) and mito-mCherry (red) and relative fluorescence intensities for the indicated linear regions measured by line scan analysis along the mito-mcherry staining (dotted lines in magnified inbox, overlay). Arrows indicate representative overlaps between M1-GFP puncta and mitochondria. Scale bar, 10 μm. (B) Representative live-cell image of HeLa cells expressing M1-GFP (green) and mito-mCherry (red). Time-lapse images of boxed area reveal interaction between M1-GFP puncta (arrows) and dynamic mitochondrion over the entire video. Time is indicated in min:sec. Scale bar, 10 μm. (C) HeLa cells were transfected with M1-GFP, fixed, and then stained for TOM20 and calreticulin to visualize mitochondria and ER, respectively. Arrows in the inbox indicate representative overlaps between M1-GFP puncta, ER, and mitochondria. Scale bar, 10 μm. (D) Line scan analysis of the intensity fluorescence along the TOM20 staining (dot line in the overlay inbox) reveals partial overlaps between mitochondria, ER, and M1-GFP. (E) Quantification of the overlap as percentage between M1-GFP and TOM20 or calreticulin and TOM20 or M1-GFP and calreticulin staining. Data are shown as mean ± SEM. One-way ANOVA Tukey’s multiple comparisons test. ∗∗∗ p < 0.005; ∗∗∗∗ p < 0.001; ns, not significant. n, number of cells analyzed. (F) Immunoblots of subcellular fractions isolated from HeLa cells, mouse brain, or mouse liver tissues. The following markers were used: IP3R3 for the ER, VDAC1, and cytochrome c (Cyt C) for mitochondria (Mp, mito pure), SigmaR1 for MAMs (MERCs), and β-tubulin for cytosol (Cyt). H: homogenate. All markers were enriched in their respective compartments. The close apposition between ER and mitochondria membranes at MAMs explained the presence of both VDAC1 and IP3R3 in these microdomains.

Article Snippet: Mouse monoclonal Ab (mAb) anti-spastin 6C6 (Cat# ab77144; WB 1:1000), rabbit polyclonal Ab (pAb) anti-β-tubulin (Cat# ab6046; WB 1:10000), mouse mAb anti-TOM20 (Cat# ab56783; WB 1:2500; IF 1:2000), rabbit pAb anti-IP3R (Cat# ab5804, PLA 1:600), mouse mAb anti-VDAC1 (Cat# ab14734, PLA 1:200) and rabbit pAb anti-VDAC1 (Cat# ab15895; WB 1:2 500; PLA 1:200) were from Abcam.

Techniques: Fluorescence, Staining, Expressing, Transfection, Western Blot, Isolation

Journal: iScience

Article Title: Spastin regulates ER-mitochondrial contact sites and mitochondrial homeostasis

doi: 10.1016/j.isci.2024.110683

Figure Lengend Snippet: Depletion of spastin increases the number of MERCs (A–C) HeLa cells were mock transfected or treated with scramble or siRNA SPAST for 72 h. Cells were then lysed or fixed and processed for WB and PLA, respectively. (A) Representative immunoblot of protein extracts for spastin and GAPDH and quantification of the band densities normalized to both GAPDH and scramble condition. (B) Cells were costained for VDAC1 (cat# ab15895) and IP3R (cat# 610312, upper panels) or labeled only for VDAC1 (bottom panels) as control. Cells were then processed for PLA as described in MM. (C) Quantification of the surface of PLA-positive spots per surface of the cell. Data are normalized to scramble condition stained only with VDAC1. (D) SH-SY5Y cells were treated twice with scramble or siRNA SPAST for 72 h and then fixed and processed for PLA. SH-SY5Y cells were cotransfected with GFP to better visualize their morphology. Cells were costained for VDAC1 (cat# ab14734) and IP3R (cat# ab5804). (E) Quantification of the surface of PLA-positive spots in GFP-positive cell per area of each cell shown in (D). Data are normalized to scramble condition. (F) Mouse embryonic fibroblasts from WT or Spg4-KO mice were fixed, costained for VDAC1 (cat# ab15895) and IP3R (cat# 610312, upper panels) or labeled only for VDAC1 (bottom panels) and processed for PLA. (G) Quantification of the surface of PLA signal per area of each cell shown in (F). Data are normalized to WT fibroblasts stained only with VDAC1. Data are shown as mean ± SEM. One-way ANOVA Tukey’ or Sidak’s multiple comparisons test. ∗ p < 0.05; ∗∗∗ p < 0.005; ∗∗∗∗ p < 0.001; ns, not significant. n, number of cells analyzed. Scale bars, 10 μm.

Article Snippet: Mouse monoclonal Ab (mAb) anti-spastin 6C6 (Cat# ab77144; WB 1:1000), rabbit polyclonal Ab (pAb) anti-β-tubulin (Cat# ab6046; WB 1:10000), mouse mAb anti-TOM20 (Cat# ab56783; WB 1:2500; IF 1:2000), rabbit pAb anti-IP3R (Cat# ab5804, PLA 1:600), mouse mAb anti-VDAC1 (Cat# ab14734, PLA 1:200) and rabbit pAb anti-VDAC1 (Cat# ab15895; WB 1:2 500; PLA 1:200) were from Abcam.

Techniques: Transfection, Western Blot, Labeling, Control, Staining

Journal: iScience

Article Title: Spastin regulates ER-mitochondrial contact sites and mitochondrial homeostasis

doi: 10.1016/j.isci.2024.110683

Figure Lengend Snippet: MERCs deregulation in spastin-lacking cells does not correlate with altered microtubule dynamics (A) HeLa cells were mock transfected or treated with scramble or siRNA SPAST for 72 h as described in MM, then their protein extracts were analyzed by immunoblot. (B) Quantification of the band densities in (A). Data are normalized to GAPDH and scramble conditions. No significant statistical difference was observed. (C–F) Spg4-KO or WT MEF were cultured, and proteins were extracted and analyzed by immunoblot (C and D) or cells were fixed and costained for acetylated (red) and tyrosinated (green) tubulin (E and F). Scale bar, 20 μm. (D) Quantification of the band densities of the immunoblot in (C). Data were normalized to both GAPDH and WT signal. (F) Ratio between acetylated and tyrosinated average fluorescence intensities measured for each cell in (E). (G) HeLa cells were treated with scramble or siRNA SPAST for 48 h, then transfected with GFP-tagged full-length mouse spastin M1 (M1-GFP) or mutated forms of the protein (M1CY-GFP, M1RC-GFP, or M1Δ-GFP), fixed after 24 h and finally processed for PLA using VDAC1 (cat# ab14734) and IP3R (cat# ab5804) antibodies. Scale bars, 10 μm. (H) Quantification of PLA surface spots in GFP-positive cells per area of each cell shown in (G). Data are normalized to scramble condition. Data are shown as mean ± SEM. One-way ANOVA Tukey’s multiple comparisons test for (B), (D), and (H), unpaired Student’s t test for (F). ∗ p < 0.05; ∗∗∗∗ p < 0.001. ns, not significant. n, number of cells analyzed.

Article Snippet: Mouse monoclonal Ab (mAb) anti-spastin 6C6 (Cat# ab77144; WB 1:1000), rabbit polyclonal Ab (pAb) anti-β-tubulin (Cat# ab6046; WB 1:10000), mouse mAb anti-TOM20 (Cat# ab56783; WB 1:2500; IF 1:2000), rabbit pAb anti-IP3R (Cat# ab5804, PLA 1:600), mouse mAb anti-VDAC1 (Cat# ab14734, PLA 1:200) and rabbit pAb anti-VDAC1 (Cat# ab15895; WB 1:2 500; PLA 1:200) were from Abcam.

Techniques: Transfection, Western Blot, Cell Culture, Fluorescence

Journal: iScience

Article Title: Spastin regulates ER-mitochondrial contact sites and mitochondrial homeostasis

doi: 10.1016/j.isci.2024.110683

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal Ab (mAb) anti-spastin 6C6 (Cat# ab77144; WB 1:1000), rabbit polyclonal Ab (pAb) anti-β-tubulin (Cat# ab6046; WB 1:10000), mouse mAb anti-TOM20 (Cat# ab56783; WB 1:2500; IF 1:2000), rabbit pAb anti-IP3R (Cat# ab5804, PLA 1:600), mouse mAb anti-VDAC1 (Cat# ab14734, PLA 1:200) and rabbit pAb anti-VDAC1 (Cat# ab15895; WB 1:2 500; PLA 1:200) were from Abcam.

Techniques: Recombinant, Bicinchoninic Acid Protein Assay, In Situ, Lysis, Sequencing, Plasmid Preparation, Software

Journal: Scientific Reports

Article Title: Nanobiopsy investigation of the subcellular mtDNA heteroplasmy in human tissues

doi: 10.1038/s41598-024-64455-0

Figure Lengend Snippet: Quad-immunofluorescence primary and secondary antibodies.

Article Snippet: VDAC1 , Mouse anti-VDAC1 (IgG2b) , ab14734 , Abcam , 1:100 , Alexa Fluor 546 Anti-Mouse IgG2b , A21143 , Life technologies , 1:200.

Techniques:

Journal: iScience

Article Title: VDAC1-interacting molecules promote cell death in cancer organoids through mitochondrial-dependent metabolic interference

doi: 10.1016/j.isci.2024.109853

Figure Lengend Snippet:

Article Snippet: Mouse-Anti-VDAC1 [N152B/23R] , Addgene , Cat# 184197-rAb; RRID: AB_2909559.

Techniques: Recombinant, Virus, Modification, Saline, Membrane, Cell Culture, Staining, Protease Inhibitor, Viability Assay, Purification, Reverse Transcription, Western Blot, Software, Imaging