Anti-VDAC1/Porin + VDAC2 + VDAC3 antibody – Mitochondrial Loading Control

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VDAC1/Porin + VDAC2 + VDAC3 antibody – Mitochondrial Loading Control (AB15895)

IHC image of VDAC1/Porin + VDAC2 + VDAC3 staining in a section of formalin-fixed paraffin-embedded normal human heart performed on a Leica Biosystems BOND® RX instrument. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab15895, 0.1 μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody. For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times. Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VDAC1/Porin + VDAC2 + VDAC3 antibody – Mitochondrial Loading Control (AB15895)

Immunohistochemical analysis of formalin fixed paraffin embedded human heart labelling VDAC1/Porin with ab15895 at a concentration of 0.1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab15895 Anti-VDAC1/Porin antibody was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-VDAC1/Porin + VDAC2 + VDAC3 antibody – Mitochondrial Loading Control (AB15895)

ab15895 staining VDAC1/Porin + VDAC2 + VDAC3 in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab15895 at 5µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• WB

Project

Western blot - Anti-VDAC1/Porin + VDAC2 + VDAC3 antibody – Mitochondrial Loading Control (AB15895)

All lanes:

Western blot - Anti-VDAC1/Porin + VDAC2 + VDAC3 antibody – Mitochondrial Loading Control (ab15895) at 1 µg/mL

Lane 1:

Hela Nuclear lysate at 20 µg

Lane 2:

Hela cell lysate at 20 µg

Lane 3:

A431 cell lysate at 20 µg

Lane 4:

Jurkat cell lysate at 20 µg

Lane 5:

HEK293 cell lysate at 20 µg

Lane 6:

Hela Nuclear lysate at 20 µg with Human VDAC1/Porin peptide (<a href='/ko/products/unavailable/human-vdac1porin-peptide-ab16131'>ab16131</a>)

Lane 7:

Hela cell lysate at 20 µg with Human VDAC1/Porin peptide (<a href='/ko/products/unavailable/human-vdac1porin-peptide-ab16131'>ab16131</a>)

Lane 8:

A431 cell lysate at 20 µg with Human VDAC1/Porin peptide (<a href='/ko/products/unavailable/human-vdac1porin-peptide-ab16131'>ab16131</a>)

Lane 9:

Jurkat cell lysate at 20 µg with Human VDAC1/Porin peptide (<a href='/ko/products/unavailable/human-vdac1porin-peptide-ab16131'>ab16131</a>)

Lane 10:

HEK293 cell lysate at 20 µg with Human VDAC1/Porin peptide (<a href='/ko/products/unavailable/human-vdac1porin-peptide-ab16131'>ab16131</a>)

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab6721'>ab6721</a>) at 1/5000 dilution

Predicted band size: 31 kDa

Observed band size: 31 kDa

true

• WB

Unknown

Western blot - Anti-VDAC1/Porin + VDAC2 + VDAC3 antibody – Mitochondrial Loading Control (AB15895)

Western blot image using the Optiblot Reducing Electrophoresis Kit - 10 x 10 cm (4-20%) (ab119220) with the
Prism Ultra Protein Ladder (ab116028) 5μl used. We recommend using our ECL substrate kit (ab65623) .
20ug of Lysate per lane and detection using ab15895 diluted to 1ug/ml.
Lane 1 : HeLa cell lysate
Lane 2 : Jurkat cell lysate
Lane 3 : A431 cell lysate
Lane 4 : HEK293 cell lysate
Lane 5 : HepG2 cell lysate.

All lanes:

Western blot - Anti-VDAC1/Porin + VDAC2 + VDAC3 antibody – Mitochondrial Loading Control (ab15895)

Predicted band size: 31 kDa

true

Exposure time: 3min

• WB

Project

Western blot - Anti-VDAC1/Porin + VDAC2 + VDAC3 antibody – Mitochondrial Loading Control (AB15895)

All lanes:

Western blot - Anti-VDAC1/Porin + VDAC2 + VDAC3 antibody – Mitochondrial Loading Control (ab15895) at 1 µg/mL

Lane 1:

Heart (Mouse) Tissue Lysate at 10 µg

Lane 2:

Kidney (Mouse) Tissue Lysate at 10 µg

Lane 3:

Western blot - Mouse skeletal muscle tissue lysate - total protein (<a href='/ko/products/tissue-lysates/mouse-skeletal-muscle-tissue-lysate-total-protein-ab29711'>ab29711</a>) at 10 µg

Lane 4:

Spinal Cord (Mouse) Tissue Lysate at 10 µg

Lane 5:

PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate at 10 µg

Lane 6:

Brain (Rat) Tissue Lysate - normal tissue at 10 µg

Lane 7:

Kidney (Rat) Whole Cell Lysate - normal tissue (<a href='/ko/products/unavailable/kidney-rat-whole-cell-lysate-normal-tissue-ab29480'>ab29480</a>) at 10 µg

Secondary

All lanes:

IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Predicted band size: 31 kDa

Observed band size: 31 kDa

false

• WB

Project

Western blot - Anti-VDAC1/Porin + VDAC2 + VDAC3 antibody – Mitochondrial Loading Control (AB15895)

All lanes:

Western blot - Anti-VDAC1/Porin + VDAC2 + VDAC3 antibody – Mitochondrial Loading Control (ab15895) at 1 µg/mL

Lane 1:

Chicken liver cell lysate at 20 µg

Lane 2:

CHO K1 cell lysate at 20 µg

Lane 3:

MDCK cell lysate at 20 µg

Lane 4:

Chicken liver cell lysate at 20 µg with Human VDAC1/Porin peptide (<a href='/ko/products/unavailable/human-vdac1porin-peptide-ab16131'>ab16131</a>)

Lane 5:

CHO K1 cell lysate at 20 µg with Human VDAC1/Porin peptide (<a href='/ko/products/unavailable/human-vdac1porin-peptide-ab16131'>ab16131</a>)

Lane 6:

MDCK cell lysate at 20 µg with Human VDAC1/Porin peptide (<a href='/ko/products/unavailable/human-vdac1porin-peptide-ab16131'>ab16131</a>)

Secondary

All lanes:

Alexa fluor goat polyclonal anti-Rabbit IgG at 1/10000 dilution

Predicted band size: 31 kDa

Observed band size: 31 kDa

true

• WB

CiteAb

Western blot - Anti-VDAC1/Porin + VDAC2 + VDAC3 antibody – Mitochondrial Loading Control (AB15895)

Western Blotting using Anti-VDAC1/Porin + VDAC2 + VDAC3 antibody – Mitochondrial Loading Control, ab15895. Publication image from Fabbri, L. et al., 2020, Theranostics, 32194829. Legend direct from paper.

The presence of VDAC1-DC in ccRCC cells decreases or abolishes ciliation. A, Triple immunofluorescence labeling and merged images with acetylatedα-tubulin (Acet.α-tubulin in red), Arl13b (in green) and DAPI (in blue). B, Electron microscopy of RCC4+pVHL cells. C, Quantitative analysis of the ciliation percentage was assessed by confocal fluorescence microscopy (n=100-300 cells). D, Both cell lines were seeded at the same density and incubated in Nx for 48h with or without serum. Percentage of ciliated cells, proliferation and FACS analysis were measured. The mean ± SEM is representative of three independent experiments carried out in duplicate. E, F and G, RCC4 cells were transfected with control siRNA (siCtl), (E) siHIF-1α, siHIF-2α and siHIF-1/2α, (F) siVDAC1 and (G) siLGMN. Cell lysates were analyzed by immunoblotting for HIF-1α, HIF-2α, VDAC1, LGMN and β- tubulin/Actin or HSP90 were used as a loading control. Quantitative analysis of the ciliation percentage was assessed by confocal fluorescence microscopy (n=100-300 cells). A * p<0.05 shows significant differences. Quantification of VDAC1 and VDAC1-δC protein levels (E). Experiments have been proceeded without serum.

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• WB

CiteAb

Western blot - Anti-VDAC1/Porin + VDAC2 + VDAC3 antibody – Mitochondrial Loading Control (AB15895)

Western Blotting using Anti-VDAC1/Porin + VDAC2 + VDAC3 antibody – Mitochondrial Loading Control, ab15895. Publication image from Fabbri, L. et al., 2020, Theranostics, 32194829. Legend direct from paper.

The presence of VDAC1-DC in ccRCC cells decreases or abolishes ciliation. A, Triple immunofluorescence labeling and merged images with acetylatedα-tubulin (Acet.α-tubulin in red), Arl13b (in green) and DAPI (in blue). B, Electron microscopy of RCC4+pVHL cells. C, Quantitative analysis of the ciliation percentage was assessed by confocal fluorescence microscopy (n=100-300 cells). D, Both cell lines were seeded at the same density and incubated in Nx for 48h with or without serum. Percentage of ciliated cells, proliferation and FACS analysis were measured. The mean ± SEM is representative of three independent experiments carried out in duplicate. E, F and G, RCC4 cells were transfected with control siRNA (siCtl), (E) siHIF-1α, siHIF-2α and siHIF-1/2α, (F) siVDAC1 and (G) siLGMN. Cell lysates were analyzed by immunoblotting for HIF-1α, HIF-2α, VDAC1, LGMN and β- tubulin/Actin or HSP90 were used as a loading control. Quantitative analysis of the ciliation percentage was assessed by confocal fluorescence microscopy (n=100-300 cells). A * p<0.05 shows significant differences. Quantification of VDAC1 and VDAC1-δC protein levels (E). Experiments have been proceeded without serum.

false

• WB

CiteAb

Western blot - Anti-VDAC1/Porin + VDAC2 + VDAC3 antibody – Mitochondrial Loading Control (AB15895)

Western Blotting using Anti-VDAC1/Porin + VDAC2 + VDAC3 antibody – Mitochondrial Loading Control, ab15895. Publication image from Hajnoczky, G. et al., 2019, Nat Commun, 31586055. Legend direct from paper.

Loss of mCa2+ uptake enhances the myofibroblast differentiation. aMcu conditional allele with LoxP sites flanking exons 5–6. b Experimental timeline for deletion of Mcu in mouse embryonic fibroblasts (MEFs). MEFs were isolated from Mcufl/fl embryos at E13.5 and infected with adenovirus encoding Cre recombinase (Ad-Cre) or the control beta-galactosidase (Ad-βgal) for 24 h. c Expression of mtCU components was examined by Western blot in Mcu−/− (Ad-Cre) and control (Ad-βgal) MEFs. MICU1 Mitochondrial Ca2+ Uptake 1, MCUR1 Mitochondrial Ca2+ Uniporter Regulator 1, MCUB Mitochondrial Ca2+ Uniporter B, EMRE Essential MCU Regulator. Mitochondrial loading controls : Voltage-dependent anion channel (VDAC) and complex III (CIII, subunit-UQCRC2) served as mitochondrial loading controls and tubulin as total lysate loading control. dMcu−/− and control were transduced with adenovirus encoding the mitochondrial calcium sensor, Mito R-GECO. 1 mM ATP was delivered to initiate IP3R-mediated Ca2+ release; n = 13 cells. e Amplitude (peak intensity–baseline). fMcu−/− and control were loaded with the Ca2+-sensitive dye Fluo-4 AM. Fluorescence was recorded during 1 mM ATP treatment; n = 15 cells Ad-βgal, n = 17 cells Ad-Cre. g Amplitude (peak intensity–baseline). h–l MEFs were treated with TGFβ or AngII for 24 h and immunofluorescence was performed by costaining withα-smooth muscle actin (α-SMA) antibody (red) and DAPI (blue); n = 3. h–j Representative images. k Percentage ofα-SMA positive cells. lα-SMA expression (fluorescence intensity). m, n Collagen gel contraction assay; n = 4 Ad-βgal, n = 5 Ad-Cre. m Representative images. n Gel contraction calculated as percent change from time 0 h. o Fold change in expression of myofibroblast genes (vs. Ad-βgal control). Col1a1 collagen type I alpha 1 chain, Col1a2 collagen type I alpha 2 chain, Col3a1 collagen type III alpha 1 chain,α-SMA (Acta2)α-smooth muscle actin, Postn periostin, Lox lysyl oxidase, Fn1 fibronectin 1, Pdgfra platelet derived growth factor receptor alpha col1a1 (n = 7), col1a2 (n = 4), col3a1 (n = 5), aSMA (n = 5), Postn (n = 9), Lox (n = 6), Fn1 (n = 6), Pdgfra (n = 6). p Cell proliferation measured by quantifying DNA content; n = 6. Ca2+ traces : solid line = mean, dashed line = SEM. Data shown as mean ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05 vs. vehicle control analyzed by ANOVA. ###p < 0.001, ##p < 0.01, #p < 0.05 vs. Ad-βgal analyzed by t-test. Scale bar = 50 µm. Also see Supplementary Fig. 1

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• WB

CiteAb

Western blot - Anti-VDAC1/Porin + VDAC2 + VDAC3 antibody – Mitochondrial Loading Control (AB15895)

Western Blotting using Anti-VDAC1/Porin + VDAC2 + VDAC3 antibody – Mitochondrial Loading Control, ab15895. Publication image from Hajnoczky, G. et al., 2019, Nat Commun, 31586055. Legend direct from paper.

Profibrotic stimuli alter mtCU gating to reduce mCa2+ uptake. a MEFs plus or minus (+/−) 12 h TGFβ were loaded with Ca2+-sensitive dye Fura-2. Fluorescence was recorded and 1 mM ATP was delivered to initiate IP3R-mediated Ca2+ release. Cytosolic Ca2+ (cCa2+) load was determined by calculating area-under-the-curve (AUC) of cCa2+ transients; n = 28 cells. b MEFs +/− 12 h TGFβ were transduced with adenovirus encoding the mitochondrial calcium (mCa2+) sensor, Mito-R-GECO. Fluorescence was recorded during 1 mM ATP treatment. mCa2+ load was determined by calculating AUC; n = 24 cells. c–g MEFs +/− 12 h TGFβ were permeabilized with digitonin in the presence of thapsigargin (SERCA inhibitor) and CGP-37157 (NCLX inhibitor) and loaded with Ca2+ sensor Fura-2 and δψ sensor JC-1 for ratiometric monitoring during Ca2+ additions. c Representative Ca2+ traces in untreated (black) and TGFβ-treated (blue) MEFs. d JC-1 derived δψ in untreated (black) and TGFβ-treated (blue) MEFs. e, f Dose response curve of mCa2+ uptake following different [Ca2+] boluses. g Kinetic parameters derived from data in panel e. h–j MEFs were treated with TGFβ and cell lysates were immunoblotted for components of the mtCU, including pore forming subunit MCU and regulatory subunits MICU1 (Mitochondrial Ca2+ Uptake 1), MCUR1 (Mitochondrial Ca2+ Uniporter Regulator 1), MCUB, and EMRE (Essential MCU Regulator), as well as OxPhos Complexes CV (ATP5A) and CIII (subunit-UQCRC2), VDAC (Voltage-dependent anion channel), and tubulin. Mitochondrial loading controls : VDAC and CIII; total lysate loading control : tubulin. Band density was normalized to CIII; n = 3. k, l MEFs and mouse adult cardiac fibroblasts (ACFs) were treated with TGFβ and Micu1 mRNA was analyzed by qPCR. n = 6. m–o MEFs were treated with AngII and cell lysates were immunoblotted for MCU, MICU1, MCUR1, MCUB, and EMRE, as well as OxPhos Complexes CV and CIII, VDAC, and tubulin. Mitochondrial loading controls : VDAC and CIII. Total lysate loading control : tubulin. Band density was normalized to CIII; n = 3. p, q MEFs and mouse ACFs were treated with AngII and Micu1 mRNA was analyzed by qPCR (n = 6). Data shown as mean ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05 vs. vehicle control analyzed by ANOVA. Also see Supplementary Fig. 2

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