Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: USP11 knockout HAP1 cell lysate (20 µg)
Lane 3: LNCaP cell lysate (20 µg)
Lane 4: HeLa cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab109232 observed at 110 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab109232 was shown to specifically react with USP11 when USP11 knockout samples were used. Wild-type and USP11 knockout samples were subjected to SDS-PAGE. ab109232 and ab8245 (loading control to GAPDH) were diluted 1/5000 and 1/2000 respectively 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 1/10 000 dilution for 1 h at room temperature before imaging.

Overlay histogram showing Jurkat cells fixed in 4% PFA and stained with purified ab109232 at a dilution of 1 in 950 (red line). The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 500. Rabbit monoclonal IgG was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line).

ab109232 (purified) at 1/300 immunoprecipitating USP11 in 10 μg HEK293 (Lanes 1 and 2, observed at 110 kDa). Lane 3 - PBS. For western blotting, a HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG was used as the secondary antibody (1/1500). Blocking buffer and concentration: 5% NFDM/TBST Dilution buffer and concentration: 5% NFDM/TBST

All lanes : Anti-USP11 antibody [EPR4346] (ab109232) at 1/5000 dilution (purified)

Lane 1 : mouse brain lysate
Lane 2 : rat brain lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit IgG (H+L) at 1000 µg

Predicted band size: 110 kDa
Observed band size: 110 kDa

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Flow cytometry analysis of 2% paraformaldehyde fixed Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling USP11 with unpurified ab109232 at 1/570 dilution (red line). Secondary antibody used is a goat anti rabbit IgG (FITC) at 1/150 dilution. The isotype control is rabbit monoclonal IgG (black line). The unlabeled control is cells without incubation with primary and secondary antibodies (blue line).

ab109232 (purified) at 1/300 immunoprecipitating USP11 in 10 μg Jurkat (Lanes 1 and 2, observed at 110 kDa). Lane 3 - PBS. For western blotting, a HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG was used as the secondary antibody (1/1500). Blocking buffer and concentration: 5% NFDM/TBST Dilution buffer and concentration: 5% NFDM/TBST

All lanes : Anti-USP11 antibody [EPR4346] (ab109232) at 1/5000 dilution (purified)

Lane 1 : HEK293 lysate at 20 µg
Lane 2 : human fetal kidney lysate

Secondary
All lanes : HRP goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 110 kDa
Observed band size: 110 kDa

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

All lanes : Anti-USP11 antibody [EPR4346] (ab109232) at 1/1000 dilution (purified)

Lane 1 : Jurkat lysate
Lane 2 : human testis lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 110 kDa
Observed band size: 110 kDa

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

All lanes : Anti-USP11 antibody [EPR4346] (ab109232) at 1/1000 dilution (unpurified)

Lane 1 : 293T cell lysate
Lane 2 : Jurkat cell lysate
Lane 3 : Human testis cell lysate
Lane 4 : Huma fetal kidney cell lysate
Lane 5 : LnCaP cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 110 kDa
Observed band size: 110 kDa