Anti-SDHB antibody [21A11AE7]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SDHB antibody [21A11AE7] (AB14714)

ab14714 staining SDHB in wild-type HEK293 cells (top panel) and SDHB knockout HEK293 cells (bottom panel). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% 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 with ab14714 at 5μg concentration and ab6046 (Rabbit polyclonal to beta Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to mouse IgG (Alexa Fluor® 488) (ab150117) at 2 μg/ml (shown in green) and a goat secondary antibody to rabbit IgG (Alexa Fluor® 594) (ab150080) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• Flow Cyt

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Flow Cytometry - Anti-SDHB antibody [21A11AE7] (AB14714)

Overlay histogram showing HEK-293 (human epithelial cell line from embryonic kidney) cells stained with ab14714 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab14714, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-SDHB antibody [21A11AE7] (AB14714)

ICC/IF image of ab14714 stained HeLa (human epithelial cell line from cervix adenocarcinoma) cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab14714, 1μg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43μM.

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-SDHB antibody [21A11AE7] (AB14714)

ab14714 staining SDHB in normal ageing human colon tissue by Immunohistochemistry (Frozen sections).

• WB

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Western blot - Anti-SDHB antibody [21A11AE7] (AB14714)

Extra bands in the mouse sample (lane 4) are due to the reaction of the IgG-specific goat anti-mouse secondary antibody with residual mouse blood in the heart tissue, as it is very difficult to entirely remove the blood from these small organs.

All lanes:

Western blot - Anti-SDHB antibody [21A11AE7] (ab14714) at 5 µg/mL

Lane 1:

Isolated mitochondria from Human heart at 5 µg

Lane 2:

Isolated mitochondria from Bovine Heart at 1 µg

Lane 3:

Isolated mitochondria from Rat heart at 10 µg

Lane 4:

Isolated mitochondria from Mouse heart at 10 µg

Lane 5:

Isolated mitochondria from HepG2 (human liver hepatocellular carcinoma cell line) cells at 20 µg

Secondary

All lanes:

Goat anti-Mouse secondary

Predicted band size: 32 kDa

Observed band size: 28 kDa,55 kDa

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• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-SDHB antibody [21A11AE7] (AB14714)

Mitochondrial localization of complex II visualized by immunocytochemistry using anti-complex II subunit 30 kDa Ip mAb 21A11 (ab14714). Cells were fixed, permeabilized and then labeled with ab14714 followed by an Alexa Fluor® 488-conjugated-goat-anti-mouse IgG2a isotype specific secondary antibody.

• WB

CiteAb

Western blot - Anti-SDHB antibody [21A11AE7] (AB14714)

Western Blotting using Anti-SDHB antibody [21A11AE7], ab14714. Publication image from Protasoni, M. et al., 2020, EMBO J, 31912925. Legend direct from paper.

Complex I and IV enzymatic deficiencies in δ4‐CYB cellsThe activities (mUnits/g of protein) of the MRC enzymes were determined by spectrophotometric kinetic measurements in WT and δ4‐CYB cells and normalized by the percentage of citrate synthase (CS) activity. Results are expressed as mean ± SD (n = 4–6 biological replicates). Unpaired Student's t‐test **P = 0.0100; ***P = 0.0002; ****P < 0.0001.Complex I in‐gel activity assays (IGA) after blue‐native gel electrophoresis (BNGE) of WT and δ4‐CYB samples solubilized with either 1.6 mg DDM/mg protein or 4 mg digitonin/mg protein. The gels were incubated in the reaction mixture for 1.5 h (lighter signals in DDM gels) or were left to continue the reaction for 24 h to obtain darker signals (DDM and Digitonin gels).BNGE, Western blot, and immunodetection, with anti‐NDUFS1 (cI), anti‐ATP5A (cV), and anti‐SDHB (cII) antibodies, of samples from the WT cybrids and from δ4‐CYB clones E (#17.3E) and B (#17.3B). Clone E was the cell line of choice for the analysis shown in panels (A and B), and all the figures hereafter.Source data are available online for this figure.

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• WB

CiteAb

Western blot - Anti-SDHB antibody [21A11AE7] (AB14714)

Western Blotting using Anti-SDHB antibody [21A11AE7], ab14714. Publication image from Wydro, M. et al., 2013, Cell Metab, 23473034. Legend direct from paper.

GRSF1 Is Required for Mitochondrial Gene Expression(A) Left panel, immunoblot analysis of 143B cells treated with control (Ctrl) or GRSF1 RNAi1. Right panel, immunoblot analysis of 143B cells infected with lentiviruses carrying an empty vector (pLKO) or a shRNA (RNAi2) against GRSF1. COX1 and COX2 are mitochondrially encoded proteins. SDHB and Tom20 are nuclear-encoded mitochondrial proteins. Actin is a nuclear-encoded cytosolic protein.(B) Acidification of the culture medium in GRSF1 RNAi1-treated 143B cells. The number of cells was the same in both conditions. Similar results were obtained in GRSF1 RNAi2-infected cells (data not shown).(C) NanoString analysis of 11 mitochondrially encoded ORFs in control (Ctrl) or GRSF1 RNAi1-treated 143B cells. RNA14 corresponds to bicistronic MTATP8-MTATP6 RNA. Data are shown as mean ± SEM (n = 3).(D) NanoString analysis of GRSF1 in control (Ctrl) or GRSF1 RNAi1-treated 143B cells. Data are shown as mean ± SEM (n = 3).(E) NanoString analysis of 11 nuclear-encoded proteins in control (Ctrl) or GRSF1 RNAi1-treated 143B cells. Data are shown as mean ± SEM (n = 3).(F) Northern blot analysis of 12S and 16S rRNA in control (Ctrl) or GRSF1 RNAi1-treated cells. TUB, tubulin.(G) Phosphorimager quantification of (E). Data are shown as mean ± SEM (n = 3).(H) 35S-labeling of mitochondrial translation of 143B cells infected with lentiviruses carrying an empty vector (pLKO) or a shRNA (RNAi2) against GRSF1.(I) Phosphorimager quantification of (H). Data are normalized to the Ponceau S intensity and are shown as mean ± SEM (n = 4).

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• WB

CiteAb

Western blot - Anti-SDHB antibody [21A11AE7] (AB14714)

Western Blotting using Anti-SDHB antibody [21A11AE7], ab14714. Publication image from Sharma, V. et al., 2020, Nat Commun, 31949167. Legend direct from paper.

Analysis of RISP steady state level in CIII.a, b BNGE analyses of digitonin-solubilized mitochondria from a liver and b kidney. ATP5A and SDHB are shown as loading controls. The individually analyzed and quantified samples were pooled together to prepare the representative blots. SC supercomplex. Error bars represent 95% CI of the mean.

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