Mouse monoclonal anti beta actin

Tumor cell lysates were obtained by lysing the tumor cells in RIPA lysis buffer mixed with a phosphatase inhibitor and a protease inhibitor (Beyotime, China), which was heated at 99°C for 15 min. An equal amount of protein (40 μg) was separated by SDS-PAGE on an 8-12% acrylamide gel for electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). Primary antibodies were diluted in skimmed milk powder prepared from TBST: rabbit monoclonal anti-STAT3 antibody, 1 : 1000 (Cell Signaling Technology, USA); rabbit monoclonal anti-pSTAT3 antibody, 1 : 1000 (Cell Signaling Technology, USA); mouse monoclonal anti-Arg-1 antibody, 1 : 500 (Santa Cruz Biotechnology, USA); Cyclin D1 (E3P5S) XP® Rabbit mAb, 1 : 1000 (Cell Signaling Technology, USA); cytochrome c (D18C7) Rabbit mAb, 1 : 1000 (Cell Signaling Technology, USA); mouse monoclonal anti-beta-actin, 1 : 5000 (Proteintech, China). Each blot was incubated with the Chemiluminescent HRP Antibody Detection Reagent (Millipore, USA) and imaged using Image Lab software (Bio-Rad, USA).

Mouse monoclonal antibody against PHEV was generated in our laboratory. Mouse monoclonal anti-beta actin was purchased from Proteintech, and rabbit polyclonal anti-SDC1 was obtained from Abcam. Horseradish peroxidase secondary anti-mouse and rabbit IgG antibodies were obtained from Proteintech. Goat anti-mouse IgG–Alexa 568 and goat anti-rabbit IgG–Alexa 488 were purchased from Cell Signaling Technology.