Lane 1: Wild-type HEK-293T cell lysate 40 ug
Lane 2: CAPN2 knockout HEK-293T cell lysate 40 ug
Lane 3: A431 cell lysate 20 ug
Lane 4: LNCaP cell lysate 20 ug
Lanes 1 - 4:Merged signal (red and green). Green - ab75994 observed at 75 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
ab75994 was shown to react with Calpain 2 in wild-type HEK-293T cells in Western blot with loss of signal observed in CAPN2 knockout cell line ab266628 (CAPN2 knockout cell lysate ab257379). Wild-type HEK-293T and CAPN2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab75994 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 ^°C at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

Lane 1:Wild-type HEK293T cell lysate (20 ug)
Lane 2:CAPN2 knockout HEK293T cell lysate (20 ug)
Lane 3:A431 cell lysate (20 ug)
Lane 4:LNCaP cell lysate (20 ug)

ab126600 was shown to specifically react with Calpain 2 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266628 (knockout cell lysate ab257379) was used. Wild-type and Calpain 2 knockout samples were subjected to SDS-PAGE. ab126600 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2.5 hours at 1 in 1000 dilution and 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.

Allele-1: 47 bp deletion in exon 4

Allele-2: 17 bp deletion in exon 4

Allele-3: 8 bp deletion in exon 4

Allele-4: 1 bp insertion in exon 4