All lanes : Anti-HAUSP / USP7 antibody [EPR4254] (ab109109) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : TRIM24 knockout HEK293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 128 kDa
Observed band size: 128 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab109109 observed at 128 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab109109 was shown to react with HAUSP / USP7 in wild-type HEK-293T cells in western blot. The band observed in knockout cell line ab266535 (knockout cell lysate ab257284) lane below 128kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HEK-293T and USP7 knockout HEK-293T 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. ab109109 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated 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.

All lanes : Anti-HAUSP / USP7 antibody [EPR4253] (ab108931) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : USP7 knockout HEK293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 128 kDa
Observed band size: 128 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab108931 observed at 128 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab108931 was shown to react with HAUSP / USP7 in wild-type HEK-293T cells in western blot. The band observed in knockout cell line ab266535 (knockout cell lysate ab257284) lane below 128kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HEK-293T and USP7 knockout HEK-293T 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. ab108931 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated 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.

Allele-1: 1 bp insertion in exon2

 

Allele-2: Insertion of the selection cassette in exon 2.

 

Representative images of USP7 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.