All lanes : Anti-NHERF-2/SIP-1 antibody [32B6] (ab151443) at 1/500 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : SLC9A3R2 knockout HEK293T cell lysate
Lane 3 : MCF7 cell lysate
Lane 4 : T-47D cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) at 1/10000 dilution

Predicted band size: 37 kDa
Observed band size: 45 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab151443 observed at 45 kDa. Red - loading control ab52901 observed at  kDa.

 ab151443 Anti-NHERF-2/SIP-1 antibody [32B6] was shown to specifically react with NHERF-2/SIP-1 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266778 (knockout cell lysate ab258685) was used. Wild-type and NHERF-2/SIP-1 knockout samples were subjected to SDS-PAGE. ab151443 and Anti-beta Tubulin [EP1331Y] - Microtubule Marker (ab52901) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) and Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Homozygous: 11 bp deletion in exon 3

Representative images of SLC9A3R2 knockout HEK293T cells, low and high confluency examples (top left and right respectively) and wild-type HEK293T cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using an EVOS M5000 microscope.