β actin mouse monoclonal antibody

Protein samples were mixed with 5× lane marker sample buffer to create a 1× final solution loading buffer and then boiled for 5 min at 95°C. Next, the proteins were separated using sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and transferred to nitrocellulose membranes (GE Healthcare, U.S.A.) using a semidry blotting apparatus. An ICAM-1 rabbit polyclonal antibody (1:1000, Abcam, U.K.), an FN rabbit polyclonal antibody (1:500; Santa Cruz Biotechnology, U.S.A.), and a β-actin mouse monoclonal antibody (1:5000; Santa Cruz Biotechnology, U.S.A.) were used as primary antibodies. The Western blots were probed with a goat anti-rabbit or anti-mouse secondary antibody conjugated with IRDye 800 (1:5000, LI-COR Biotechnology, U.S.A.). Blotted proteins were detected and quantified using an Odyssey Infrared Imaging system (LI-COR Biotechnology, U.S.A.).

Western blot analysis was performed as previously described else where (Wang et al., 2017b (link), 2018 (link)). Granulosa cells were collected after treatment for 48 h and lysed in RIPA buffer (catalog number: 89900; ThermoFisher, Rochford, IL, USA), then denatured by boiling for 5 min with bromophenol blue and frozen at −80 °C. The proteins were separated by 12% polyacrylamide gel electrophoresis, and then transferred to polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). Firstly, the membrane were incubated with primary antibody: Bcl2 mouse monoclonal antibody (1:500, sc-7382; Santa Cruz, Dallas, TX, USA), Bax mouse monoclonal antibody (1:500, sc-20067; Santa Cruz, Dallas, TX, USA), caspase-3 rabbit polyclonal antibody (ab13847; Abcam, California, USA) and β-actin mouse monoclonal antibody (1:1000, SC-47778; Santa Cruz, Dallas, TX, USA). Later, the membrane was detected by incubation with HRP labeled goat anti-rabbit secondary antibody (SC-2054; Santa Cruz, Dallas, TX, USA) or goat anti-mouse secondary antibody (1:5000; SC-2005; Santa Cruz, Dallas, TX, USA), respectively. Finally, membranes was incubated with the Clarity Western ECL kit (catalog number: 170-5060; Bio-Rad Laboratories, Hercules, CA, USA), and scanned in a ChemiDocXRS chemiluminescent imaging system (Bio-Rad, Hercules, CA, USA).

Western blot were carried out following the procedure described in our previous study [9] (link), and primary antibodies included 1∶500 FIS1 rabbit monoclonal antibody (Sino Biological Inc, Beijing, China) and 1∶2000 β-actin mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA).