Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Phospholipase C gamma 1/PLC-gamma-1 knockout HAP1 cell lysate (20 µg)
Lane 3: HepG2 cell lysate (20 µg)
Lane 4: Mouse brain tissue lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab41433 observed at 160 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab41433 was shown to recognize Phospholipase C gamma 1/PLC-gamma-1 when Phospholipase C gamma 1/PLC-gamma-1 knockout samples were used, along with additional cross-reactive bands. Wild-type and Phospholipase C gamma 1/PLC-gamma-1 knockout samples were subjected to SDS-PAGE. ab41433 diluted to 1/1000 and ab181602 (loading control to GAPDH) diluted to 1/10,000 and incubated overnight at 4°C. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD)  preadsorbed (ab216777) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

Western blot analysis of Phospholipase C gamma 1/PLC-gamma-1 immunoprecipitates from human jurkat cells untreated (lanes 1 & 3) or treated with pervanadate (1 mM) for 30 min (lanes 2, 4, 5 & 6). Immunoprecipitation was performed with monoclonal anti-Phospholipase C gamma 1/PLC-gamma-1 (ab41433). The blots were probed with anti- Phospholipase C gamma 1/PLC-gamma-1 (lanes 1 & 2) and anti-Phospholipase C gamma 1/PLC-gamma-1 (Tyr-775) (lanes 3-6). The latter antibody was used in the presence of phospho- Phospholipase C gamma 1/PLC-gamma-1 (Tyr-775) peptide (lane 5), or a non-specific phosphotyrosine peptide (lane 6).

Overlay histogram showing Jurkat cells stained with ab41433 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab41433, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Jurkat cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.