All lanes : Anti-Lamin B Receptor/LBR antibody [BBmLBR 12.F8] (ab232731) at 1 µg/ml

Lane 1 : Wild-type HEK-293 whole cell lysate
Lane 2 : LBR knockout HEK-293 whole cell lysate
Lane 3 : Wild-type MEF-1 whole cell lysate
Lane 4 : LBR knockout MEF-1 whole cell lysate

Lysates/proteins at 20 µg/ml per lane.

Secondary
All lanes : Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) at 1/20000 dilution

Performed under reducing conditions.

Observed band size: 58 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab232731 observed at 58 kDa. Red - loading control, ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) observed at 37kDa.

ab232731 was shown to react with Lamin B Receptor (LBR) in wild-type HEK-293 and MEF-1 cells in western blot. Loss of signal was observed when LBR knockout samples were used. Wild-type and LBR knockout (ab257503) HEK-293 cell lysates  and wild-type and LBR knockout MEF-1 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk before incubation with ab232731 and ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) overnight at 4°C at 1 µg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

LBR knockout MEF-1 samples were kindly provided by the Brian Burke laboratory, A-Star Institute, Singapore.

All lanes : Anti-Lamin B Receptor/LBR antibody [E398L] (ab32535) at 1/500 dilution

Lane 1 : Wild-type HEK-293 cell lysate
Lane 2 : LBR knockout HEK-293 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Observed band size: 58 kDa why is the actual band size different from the predicted?

Lanes 1 - 2: Merged signal (red and green). Green - ab32535 observed at 58 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab32535 was shown to react with Lamin B Receptor/LBR in wild-type HEK-293 cells in western blot. Loss of signal was observed when LBR knockout lysate ab257503 was used. Wild-type and LBR knockout HEK-293 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk before incubation with ab32535 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 500 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Homozygous: 1 bp insertion in exon 2

Representative images of LBR 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.