Recombinant Anti-STAT3 (phospho Y705) antibody [EP2147Y] (ab76315)

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue labeling STAT3 (phospho Y705) with purified ab76315 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

NEAT1 influences STAT3 binding to endocytosis-related genes. The siNEAT1 and siCTRL cells were collected for ChIP assays to analyse the relative fold enrichment of the CAV2 (a), TGFB2 (b), or TGFBR1 promoter (c) with anti-STAT3 or RNAP II antibodies. The data points represent the mean values determined from three independent experiments. The data are presented as the mean ± SD. d The U251 cells were fixed and incubated with anti-STAT3 (red), anti-H3K27Ac (green) or anti-H3K27Cro antibodies (green) before the confocal analysis. The intensity plots for the red and green channels were analysed with the ImageJ software. Scale bars 10 μm. e After transfection with the siNEAT1v2 or negative control siRNA, the U251 cells were fixed and incubated with anti-STAT3 (red) or anti-H3K27Ac antibodies (green) before the confocal analysis. The intensity plots for the red and green channels were analysed with the ImageJ software. Scale bars 10 μm. f After 24 h of incubation with 80 µm crotonyl-CoA or the mock control, the U251 cell lysates were harvested and subjected to an immunoprecipitation assay with anti-STAT3 or anti-IgG antibodies. The retrieval of STAT3, H3K27Ac, H3K27Cro+ and Histone H3 by endogenous STAT3 and IgG was measured by western blotting. *p < 0.001

Immunocytochemistry/Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) +/- IFN-α (50 ng/mL, 5 minutes) cells labelling STAT3 (phospho Y705) with ab76315 at 1/500 (4.3 μg/mL). Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1500) was used as the secondary antibody. DAPI (blue) was used as a nuclear counterstain.

Immunohistochemical analysis of formalin-fixed, paraffin-embedded rat colon tissue stained for STAT3 using ab76315 at 1/1000 dilution followed by a Horse radish peroxidase conjugated Goat anti-Rabbit secondary antibody at 1/400 dilution.

Antigen Retrieval: Heat mediated - Heat mediated - Buffer/Enzyme Used: pH 9.0 EDTA

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Immunohistochemical analysis of formalin-fixed, paraffin-embedded mouse colon tissue stained for STAT3 using ab76315 at 1/1000 dilution followed by a Horse radish peroxidase conjugated Goat anti-Rabbit secondary antibody at 1/400 dilution.

Antigen Retrieval: Heat mediated - Buffer/Enzyme Used: pH 9.0 EDTA 70C 2hr.

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse spleen tissue labeling STAT3 (phospho Y705) with ab76315 at 1/100 (nuclear staining). Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody. Counterstained with hematoxylin.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon carcinoma tissue labeling STAT3 (phospho Y705) with unpurified ab76315 at 1/100. Heat mediated antigen retrieval was perfomed with citrate buffer (pH 6) 

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized A431 (Human epidermoid carcinoma epithelial cell) treated with 100ng/mL EGF for 10min (Red) / Untreated control (Green) cells labelling STAT3 with ab76315 at 1/500 dilution (0.1µg) (Red) and Green 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.

Dot blot analysis of STAT3 single phospho peptide pY705 (lane 1) and STAT3 non-phospho peptide (lane 2) with ab76315 at 1/1000. Blocking and dilution buffer was 5% NFDM/TBST. The secondary antibody used was ab97051 peroxidase conjugated Goat Anti-Rabbit IgG, (H+L) at 1/100,000.

Immunohistochemical analysis of paraffin-embedded human thyroid carcinoma tissue using  untreated (left) or alkaline phosphatase-treated (right) labeling STAT3 (phospho Y705) with ab76315 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H& L (HRP) (ab97051) at 1/500 dilution. Counter stained with Hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. 

ab76315 (purified) at 1/30 immunoprecipitating STAT3 (phospho Y705) in A431 (Human epidermoid carcinoma cell line) cell lysate treated with EGF. For western blotting, a peroxidase-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/1000).

Blocking/Dilution buffer: 5% NFDM/TBST.