Recombinant Anti-STAT3 (phospho Y705) antibody [EPR23968-52] (ab267373)

Blocking and diluting buffer and concentration: Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.

Lanes 1-4: Merged signal (red and green). Green - ab267373 observed at 88 kDa. Red - loading control ab8245 (Mouse monoclonal [6C5] to GAPDH) observed at 36 kDa.

Lanes 1-2: ab267373 Anti-STAT3 (phospho Y705) antibody [EPR23968-52] was shown to specifically react with STAT3 in wild-type serum starved and then IFN alpha treated HeLa cells. Loss of signal was observed when serum starved and then IFN alpha treated knockout cell line ab255436 (knockout cell lysate ab263797) was used. Wild-type and STAT3 knockout samples were subjected to SDS-PAGE. ab267373 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4°C overnight at 1 in 1000 dilution and 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 10000 dilution for 1 hour at room temperature before imaging.

Lysates loaded onto lanes 3-4 were made freshly and used in WB immediately to minimize protein degradation.

Dot blot analysis of STAT3 (phospho Y705) using ab267373 at 1/1000 followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1:100,000 dilution.

Lane 1: STAT3 phospho Y705 peptide (aa700-710)

Lane 2: Unmodified STAT3 peptide (aa698-710)

Exposure time: 3 minutes.

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded human endometrial carcinoma tissue labeling STAT3 (phospho Y705) with ab267373 at 1/500 dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human endometrial carcinoma without alkaline phosphatase treatment (image A) is observed. No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab267373 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 100% Methanol-permeabilized STAT3 KO HeLa cells labelling STAT3 (phospho Y705) with ab267373 at 1/500 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green).

Confocal image showing increased nuclear staining in parental HeLa cells treated with IFN-alpha (50 ng/ml) for 30 min, and no staining in STAT3 knockout HeLa cells treated with IFN-alpha (50 ng/ml) for 30 min.

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Jurkat (Human T cell leukemia T lymphocyte) treated with 50 ng/ml IFN-alpha for 30min (Red)/ Untreated control (Green) cells labelling STAT3 (phospho Y705) with ab267373 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077 at 1/2000 dilution was used as the secondary antibody.

STAT3 (phospho Y705) was immunoprecipitated from 0.35 mg Jurkat (human t cell leukemia cell line from peripheral blood) treated with 50 ng/ml IFN alpha for 30 minutes, whole cell lysate 10 µg with ab267373 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab267373 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Jurkat treated with 50 ng/ml IFN alpha for 30 minutes, whole cell lysate 10 µg

Lane 2: ab267373 IP in Jurkat treated with 50 ng/ml IFN alpha for 30 minutes whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab267373 in Jurkat treated with 50 ng/ml IFN alpha for 30 minutes whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 15 seconds.

Chromatin was prepared from HepG2 (starved overnight) treated with IL-6 cells and HepG2 (starved overnight) non-treated according to the Abcam Dual-X-ChIP protocol*.

Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.

The ChIP was performed with 25 µg of chromatin, 5 µg of ab267373 (red), or 5 µg of rabbit normal IgG ab172730 (gray) and 25 µl of Protein A/G Dynabeads. The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
Primers are purchased from competitor.
*http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

IL-6 treatment induces phosphorylation of STAT3 at Tyr705 (PMID: 28676732).

Lysates were made freshly and used in WB immediately to minimize protein degradation.

Exposure time: 26 seconds.

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

IFN alpha treatment induces phosphorylation of STAT3 at Tyr705 (PMID: 21957129).

Lysates were made freshly and used in WB immediately to minimize protein degradation.

Exposure time: 3 minutes.

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling STAT3 (phospho Y705) with ab267373 at 1/500 dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human tonsil without alkaline phosphatase treatment (image A) is observed. No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab267373 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.