Phosphor stat3 ser727

Cells were lysed 7 h after Stattic treatment, 6 h after WP1066 treatment or 24 h after irradiation with a cold RIPA buffer and protein concentration was determined using a Bradford assay (Bio-rad). An equal quantity of protein samples was separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-rad). Membrane was blocked with Phosphate-buffered saline (PBS 1X) containing 0.1% Tween 20 (Sigma) and 5% non-fat dry milk. Primary antibodies were incubated overnight at 4°C (Stat3 1:1250 (#9136); phosphor-Stat3 Tyr705 1:500 (#9131); phosphor-Stat3 Ser727 1:500 (#9136) Cell Signaling Technology; Actin 1:5000 (#ab3280) Abcam). Secondary antibodies were incubated for 1h 30 at room temperature (Anti-mouse HRP-linked 1:2000 (#7076); Anti-rabbit HRP-linked 1:2000 (#7074) Cell Signaling Technology). Immunoblotting signals were detected using an enhanced chemiluminescence method (Clarity^™ Western ECL Substrate, Bio-rad) with Luminescent Image Analyzer LAS-3000 (FUJIFILM). Densitometry analyses were performed using ImageJ software (imagej.nih.gov/ij/). Relative amounts of pY705 and pS727 were normalized to total Stat3 and Actin.

MDA-MB-231 cells were incubated with 12b at indicated concentrations, then washed with PBS and disrupted on ice for 40 min by a loading buffer, and boiled for 15 min.
The protein concentration of lysates was quantified by using a BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of protein were separated using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and then transferred to nitrocellulose membranes. The membranes were blotted with primary antibodies overnight at 4 °C followed by HRP-conjugated secondary antibodies for 1 h at room temperature. Proteins on the membranes were visualized with enhanced chemiluminescence by using chemiluminescence detection reagents.
Antibodies to detect Cleaved poly-(ADP-ribose) polymerase (C-PARP), Caspase 8, Cleaved Caspase 3 (C-Cas3), Survivin, γ-H2AX, Bcl-2, Mcl-1, P21, Akt, phosphor-Akt (Ser473), ERK1/2, phosphor-ERK1/2 (pT185/pY187), Stat3, phosphor-Stat3 (Ser727), PDGFR, FGFR1, VEGFR, EGFR, Src, and phosphor-Src (Tyr416) were purchased from Cell Signaling Technology (Boston, MA, USA). TfR1 antibody, the primary antibody β-Tubulin, and the secondary antibodies were purchased from Sangon Biotech (Shanghai, China).