All lanes : Anti-PTBP1 antibody [EPR9048(B)] (ab133734) at 1/10000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : PTBP1 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 57 kDa
Observed band size: 57 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab133734 observed at 57 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab133734 was shown to react with PTBP1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265155 (knockout cell lysate ab257614) was used. Wild-type HeLa and PTBP1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab133734 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of PTBP1 in paraffin embedded Human breast carcinoma tissue stained with ab133734 at a 1/100 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

All lanes : Anti-PTBP1 antibody [EPR9048(B)] (ab133734) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : PTBP1 knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 57 kDa

Lanes 1 - 2: Merged signal (red and green). Green - ab133734 observed at 57 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab133734 was shown to recognize PTBP1 in wild-type HAP1 cells as signal was lost at the expected MW in PTBP1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and PTBP1 knockout samples were subjected to SDS-PAGE. Ab133734 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/10000 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 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-PTBP1 antibody [EPR9048(B)] (ab133734) at 1/10000 dilution

Lane 1 : Human fetal liver lysate
Lane 2 : Jurkat cell lysate
Lane 3 : Daudi cell lysate
Lane 4 : A549 cell lysate
Lane 5 : BxPC3 cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 57 kDa

Immunofluorescent staining of PTBP1 in A549 cells, using ab133734 at a 1/250 dilution.

Flow cytometry analysis of A549 (Human lung carcinoma epithelial cell) cells labeling PTBP1 (red) with purified ab133734 at a 1/2000 dilution (1ug/mL). Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (Black) (ab172730). Blue (unlabeled control) - Cell without incubation with primary antibody and secondary antibody (Blue).