All lanes : Anti-Calcineurin A antibody [EP1669Y] (ab52761) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : PPP3CA knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 59 kDa
Observed band size: 59 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab52761 observed at 59 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab52761 was shown to react with Calcineurin A in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265130 (knockout cell lysate ab257181) was used. Wild-type HeLa and PPP3CA 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. ab52761 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 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.

Overlay histogram showing HeLa cells stained with ab52761 (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 (ab52761, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Calcineurin A knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: SH-SY5Y cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab52761 observed at 60 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab52761 was shown to specifically react with Calcineurin A when Calcineurin A knockout samples were used. Wild-type and Calcineurin A knockout samples were subjected to SDS-PAGE. ab52761 and ab8245 (loading control to GAPDH) were both diluted 1/10000 and incubated overnight at 4°C. 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 1/10000 dilution for 1 hour at room temperature before imaging.

Anti-Calcineurin A antibody [EP1669Y] (ab52761) at 1/10000 dilution + Hela cell lysate at 10 µg

Secondary
Goat anti-rabbit HRP labeled at 1/2000 dilution

Predicted band size: 59 kDa
Observed band size: 59 kDa