All lanes : Anti-PDHA1 antibody [EPR11098] (ab168379) at 1/1000 dilution

Lane 1 : Wild-type HeLa whole cell lysate
Lane 2 : PDHA1 knockout HeLa whole cell lysate
Lane 3 : HEK-293 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 43 kDa
Observed band size: 43 kDa

Lanes 1 - 3: Merged signal (red and green). Green - ab168379 observed at 43 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab168379 was shown to recognize PDHA1 in wild-type HeLa cells as signal was lost at the expected MW in PDHA1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and PDHA1 knockout samples were subjected to SDS-PAGE. Ab168379 and ab130007 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-PDHA1 antibody [EPR11098] (ab168379) at 1/2000 dilution

Lane 1 : HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates
Lane 2 : Mouse brain lysates
Lane 3 : Rat brain lysates
Lane 4 : Mouse kidney lysates
Lane 5 : Rat kidney lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 43 kDa
Observed band size: 43 kDa

Blocking and diluting buffer: 5% NFDM/TBST

Immunocytochemistry/ Immunofluorescence analysis of HT-29 (Human colorectal adenocarcinoma epithelial cell) cells labeling PDHA1 with Purified ab168379 at 1:100 dilution (3.5 μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG (Alexa Fluor^® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

ab168379 (purified) at 1:20 dilution (2ug) immunoprecipitating PDHA1 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate.
Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug
Lane 2 (+): ab168379 & HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab168379 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Flow Cytometry analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling PDHA1 with purified ab168379 at 1:40 dilution (10 ug/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilized with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat kidney tissue sections labeling PDHA1 with Purified ab168379 at 1:200 dilution (1.76 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, pH 9.0. Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thyroid carcinoma tissue sections labeling PDHA1 with Purified ab168379 at 1:200 dilution (1.76 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, pH 9.0. Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue sections labeling PDHA1 with Purified ab168379 at 1:200 dilution (1.76 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, pH 9.0. Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Immunocytochemistry/Immunofluorescence analysis Jurkat (human acute T cell leukemia) labelling PDHA1 with purified ab168379 at 1/500. Cells were fixed with 100% methanol. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (Ab150077). Nuclei counterstained with DAPI (blue).

Control: PBS only

All lanes : Anti-PDHA1 antibody [EPR11098] (ab168379) at 1/1000 dilution (unpurified)

Lane 1 : Human fetal kidney lysate
Lane 2 : A549 lysate
Lane 3 : Jurkat lysate
Lane 4 : HepG2 lysate
Lane 5 : HeLa lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 43 kDa

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling PDHA1 with unpurified ab168379 at 1/100 dilution. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue labeling PDHA1 with unpurified ab168379 at 1/100 dilution. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Immunofluorescent analysis of HepG2 cells labeling PDHA1 with unpurified ab168379 at 1/100 dilution.

Flow cytometric analysis of permeabilized Jurkat cells labeling PDHA1 (red) with unpurified ab168379 at 1/10 dilution, or a rabbit IgG (negative) (green).

Detection of PDHA1 by Western Blot of Immunprecipitate. 293T cell lysate immunoprecipitated using unpurified ab168379 at 1/10 dilution; HRP-conjugated anti-rabbit IgG preferentially detecting the non-reduced form of rabbit IgG.