Anti p stat3 tyr 705 antibody

Lysates containing 100 μg protein were mixed with loading buffer with 5% β-mercaptoethanol and heated for 5 min at 100°C. The protein samples were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes by semi-dry blotting. SDS-PAGE was performed via standard procedures according to the manufacturer’s instructions (Invitrogen Life Technologies, Carlsbad, CA, USA). Membranes were incubated in blocking buffer [Tris-buffered saline (TBS), 0.1% Tween 20 and 5% low-fat dry milk] for 1 h at room temperature, followed by hybridization with anti-p-STAT3 (tyr-705) antibody (1:1,000 dilution; Cell Signaling Technology, Inc.), anti-STAT3 antibody (1:1,000 dilution; Cell Signaling Technology, Inc.) and anti-β-actin antibody (internal control; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 4°C overnight. Following three washes in TBS/0.1% Tween 20, the membranes underwent hybridization with a horseradish peroxidase-conjugated secondary antibody rabbit IgG (1:5,000 dilution; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Following extensive washing in TBS/0.1% Tween 20, signals were detected by electrogenerated chemiluminescence techniques using SuperSignal West Pico Chemilumiscent Substrate (Thermo Fisher Scientific).

The tissue microarray (TMA) collected 55 glioma cases, in which 2 cases were of non‐tumor (cortical dysplasia), 2 cases of Grade I glioma, 12 cases of Grade II glioma, 12 Grade III, and 27 cases of GBM (Grade IV) (Figure S1 and Table S1). The procedure of immunohistochemistry (IHC) was performed as previously described.³⁵ Primary antibodies: Anti‐IKBKE antibody (Cell Signaling, USA), Anti‐pSTAT3 (Tyr705) antibody (Cell Signaling, USA) and Anti‐CD274 antibody (Abcam, USA) were incubated with a dilution of 1:100. Secondary antibodies: HRP‐labeled goat anti‐mouse IgG and goat anti‐rabbit IgG were obtained from ZSGB‐Bio, China. The IHC images were observed and acquired though a light microscope (OLYMPUS, Japan).

ChIP was performed as described previously [18 (link)]. Briefly, 1 × 10⁸ AGS cells treated with ATCC43504 multiplicity of infection (MOI) of 100 in serum free RPMI1640 medium for 0.5 hr were crosslinked with 1% formaldehyde for 10 minutes at room temperature and quenched by glycine. After cell lysis, the chromatin was fragmented into 100 - 500 bp by Bioruptor Sonicator (Diagenode, Denville, NJ) and protein-DNA complexes were immunoprecipitated (IP) by 5 ug anti-pSTAT3 (Tyr 705) antibody (Cell Signaling Technology, Beverly, MA) or anti-IgG antibody (Sigma-Aldrich) Dynal magnetic bead (Invitrogen) mix on rotator at 4°C overnight. After washing and reversal of crosslinks, the IP and input DNA were purified and subjected to PCR.