All lanes : Anti-STAT3 antibody [9D8] (ab119352) at 1/1000 dilution

Lane 1 : U2OS lysate from non-targeting control
Lane 2 : U2OS lysate from STAT3 SMART pool siRNA transfected

Lysates/proteins at 25 µg per lane.

Secondary
All lanes : goat anti-mouse-HRP at 1/20000 dilution

Developed using the ECL technique.

Predicted band size: 88 kDa



4-20% Tris-HCl polyacrylamide gel

Immunofluorescent analysis of STAT3 using ab119352 at 1/100 dilution (shown in green) in HeLa cells (formalin fixed cells permeabilized with 0.1% Triton X-100) using a DyLight 488 goat-anti-mouse secondary antibody (ab96879) at 1/400 dilution. Nuclei (blue) were stained with Hoechst 33342 dye.

Overlay histogram showing HeLa cells stained with ab119352 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween 20 for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab119352, 0.1μg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG (ab37355, 0.1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

All lanes : Anti-STAT3 antibody [9D8] (ab119352) at 1/5000 dilution

Lane 1 : HepG2 cell lysate
Lane 2 : 293T cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : A431 cell lysate
Lane 5 : U2OS cell lysate
Lane 6 : MCF7 cell lysate
Lane 7 : A549 cell lysate
Lane 8 : K562 cell lysate
Lane 9 : COS7 cell lysate
Lane 10 : NIH3T3 cell lysate
Lane 11 : C2C12 cell lysate
Lane 12 : NRK cell lysate

Lysates/proteins at 25 µg per lane.

Secondary
All lanes : goat anti-mouse-HRP at 1/20000 dilution

Developed using the ECL technique.

Predicted band size: 88 kDa



4-20% Tris-HCl polyacrylamide gel

ab119352, at 1/1600 dilution, staining STAT3 in Human pancreas tissue by Immunohistochemistry. Detection was performed using a goat anti-mouse HRP secondary antibody followed by colorimetric detection using DAB substrate. Tissues were counterstained with hematoxylin.

Anti-STAT3 antibody [9D8] (ab119352) at 1/1000 dilution + HepG2 total lysate at 25 µg

Secondary
goat anti-mouse-HRP at 1/20000 dilution

Developed using the ECL technique.

Predicted band size: 88 kDa



4-20% Tris-HCl polyacrylamide gel

ab119352, at 1/1600 dilution, staining STAT3 in Human brain tumor tissue by Immunohistochemistry. Detection was performed using a goat anti-mouse HRP secondary antibody followed by colorimetric detection using DAB substrate. Tissues were counterstained with hematoxylin.

Immunoprecipitation of STAT3 was performed on HepG2 cells. The antigen:antibody complex was formed by incubating 750µg whole cell lysate with 2µg of ab119352 overnight at 4°C. The immune-complex was captured, washed extensively and proteins eluted with 5X Reducing Sample Loading Dye. Samples were resolved on a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to PVDF membrane and blocked with 5% Milk/TBS-0.1% Tween for at least 1 hour. Membranes were then probed with ab119352 at a dilution of 1/5000 overnight at 4°C. Membranes were washed in TBST and probed with IP detection reagent at a dilution of 1/2000 for at least one hour. Membranes were washed and chemiluminescent detection was performed.

ab119352, at 1/1600 dilution, staining STAT3 in Human colon cancer tissue by Immunohistochemistry. Detection was performed using a goat anti-mouse HRP secondary antibody followed by colorimetric detection using DAB substrate. Tissues were counterstained with hematoxylin.

ab119352, at 1/1600 dilution, staining STAT3 in Human stomach cancer tissue by Immunohistochemistry. Detection was performed using a goat anti-mouse HRP secondary antibody followed by colorimetric detection using DAB substrate. Tissues were counterstained with hematoxylin.