Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: STK3/MST-2 knockout HAP1 cell lysate (20 µg)
Lane 3: NIH/3T3 cell lysate (20 µg)
Lane 4: 293T cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab52641 observed at 56 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab52641 was shown to specifically react with STK3/MST-2 when STK3/MST-2 knockout samples were used. Wild-type and STK3/MST-2 knockout samples were subjected to SDS-PAGE. ab52641 and ab8245 (loading control to GAPDH) were diluted 1/10 000 and 1/2000 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.

ab52641 staining STK3/MST-2 in wild-type HAP1 cells (top panel) and STK3/MST-2 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab52641 at 5μg/ml concentration and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Immunohistochemical staining of paraffin embedded human breast carcinoma with purified ab52641 at a working dilution of 1/50. The secondary antibody used is ab97051, a goat anti-rabbit IgG (H&L) at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

NIH/3T3 (Mouse embryonic fibroblast) cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol.  The primary antibody (ab52641) was used at a 1:70 dilution (1 μg) (red).  A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at a 1:2000 dilution.  A Rabbit monoclonal IgG (ab172730) (black) was used as an isotype control. Cells without incubation with primary antibody and secondary antibody were used as an unlabelled control (blue).  

ab52641 (purified) at 1/50 immunoprecipitating STK3/MST-2 in 10 μg C6 cell lysate (Lanes 1 and 2, observed at 56 kDa). Lane 3 - Rabbit monoclonal IgG (ab172730). For western blotting, HRP Veriblot for IP (ab131366) was used for detection (1/10 000). Blocking buffer and concentration: 5% NFDM/TBST Dilution buffer and concentration: 5% NFDM/TBST

Confocal image showing cytoplasmic staining of STK3/MST-2 in NIH/3T3 (mouse embryonic fibroblast) cells using ab52641 . The cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The cells were then incubated with ab52641 at 1/70 dilution and counterstained with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/100 dilution (red). Goat anti Rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody at 1/1000 dilution (green). Nuclei counterstained with DAPI (blue).

All lanes : Anti-STK3/MST-2 antibody [EP1466Y] (ab52641) at 1/50000 dilution (purified)

Lane 1 : C6 whole cell lysate
Lane 2 : NIH/3T3 whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 56 kDa
Observed band size: 56 kDa

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

All lanes : Anti-STK3/MST-2 antibody [EP1466Y] (ab52641) at 1/50000 dilution (purified)

Lane 1 : HEK293 whole cell lysate
Lane 2 : HeLa whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 56 kDa
Observed band size: 56 kDa

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST