Rabbit anti survivin

Cells were lysed in ice-cold radio-immunoprecipitation (RIPA) buffer containing protease inhibitor and phosphatase inhibitor cocktail (Alfa Aesar, Stoughton, MA, USA). Protein was quantified using the Bradford Assay (BioRad, Hercules, CA, USA). Proteins (20–30 μg) were resolved in SDS-polyacrylamide gels (10%) and transferred to PVDF membranes using a Tris-glycine buffer system. Membranes were blocked with 5% non-fat dry milk in 0.1% Tween20 in TBS (TBST) for 1 h at room temperature followed by probing with primary antibodies and gently rocking overnight at 4 °C. Antibodies used for immunoblotting were Mouse anti-E-cadherin (Cell Signaling, Danvers, MA, USA), Rabbit anti-N-cadherin (Cell Signaling), Rabbit anti-vimentin (Protein Technologies, Tucson, AZ, USA), Rabbit anti-survivin (Cell Signaling), Rabbit anti-Lasp1 (Cell Signaling), Rabbit anti-BMI1 (Cell Signaling), and Mouse anti-β-actin (BD Biosciences, San Jose, CA, USA). The next day, the blots were washed with TBST three times, 5 min each time. They were incubated with secondary antibodies (1:2000) at room temperature for 1 h. Chemiluminescent signals were detected with ECL™ prime (Thermo Fisher Scientific, Waltham, MA, USA) using the Biorad ChemiDoc system. If necessary, blots were stripped with ECL Stripping Buffer (Li-Cor, Lincoln, NB, USA) following the manufacturer’s protocol. Bands were quantified using ImageJ.

Cells were homogenized in cell lysis buffer (#9803, Cell Signaling Technology, Danvers MA) with protease inhibitor mixture (#11836170001, Roche). Protein samples were prepared in X2 loading buffer (#161-0737, BioRad, Portland ME) with βME following the manufacturer’s instruction. Protein was loaded and separated by electrophoresis and then transferred onto PVDF membranes (Bio-Rad). Blots were probed with the following primary antibodies: rabbit anti-survivin (#2808, Cell Signaling Technology), anti-FLAG (#8146, Cell Signaling Technology), mouse anti-ACTIN (#3700, Cell Signaling Technology), rabbit anti-USP9X (#5751, Cell Signaling Technology). All band intensities were determined using ImageJ and normalized to ACTIN signal.

Rabbit anti-phosphorylated (anti-phospho)-STAT3 at tyrosine 705 (Tyr705) and serine 727 (Ser727), rabbit anti-STAT3, rabbit anti-survivin, rabbit anti-Bcl-2, rabbit anti-Mcl-1, rabbit anti-β-actin, and anti-rabbit HRP-conjugated IgG were purchased from Cell Signaling Technology. Anti-rabbit fluorescein isothiocyanate (FITC)-conjugated IgG was purchased from Santa Cruz Biotechnology (Dallas, TX, US).

Cells were seeded into 6-well dishes, treated with 20 µM Bpep or Dpep for 72 h and processed for Western immunoblotting and imaging as previously described [33 (link)]. Antibodies used were: rabbit anti-survivin (Cell Signaling Technology, Danvers, MA, USA #2808), rabbit anti-MCL-1 (Cell Signaling Technology, Danvers, MA, USA #94296s), rabbit anti-BCL-2 (Cell Signaling Technology, Danvers, MA, USA #15071s) and mouse anti-ACTIN (Cell Signaling Technology, Danvers, MA, USA #3700).