Bcl 2 mouse monoclonal antibody

To test binding of Beclin 1 to Bcl-2, co-immunoprecipitation was performed using a mouse monoclonal Bcl-2 antibody (Santa Cruz Biotechnology, CA, USA) and immunoblotted using the mouse monoclonal Bcl-2 antibody and a mouse monoclonal Beclin 1 antibody (Santa Cruz Biotechnology, CA, USA), respectively, as described [57 (link),58 (link)].

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).

The Bax mouse monoclonal antibody, Bcl-2 mouse monoclonal antibody, Fgl2 mouse monoclonal antibody, Na⁺/K⁺-ATPase α rabbit polyclonal antibody and β-actin were purchased from Santa Cruz Biotechnology. Rabbit anti-TLR4, p38 MAPK rabbit polyclonal antibody was purchased from Beijing Zhongsha Jinqiao Company. Tumour necrosis factor (TNF)-α ELISA was purchased from Shanghai Senxiong Technology Company. Apoptosis of TUNEL cell detection kit was purchased from Promega. STZ was from Sigma; pGCSIL–Fgl2, pGC–LV (left ventricular), pHelper 1.0 and pHelper are provided by Jikai Gene Company.