Rabbit anti bcl 2 antibody

Proteins were extracted from liver IRI tissues and cultured with BMDM using RIPA Lysis Buffer (Beyotime, Shanghai, China) for Western blotting.
Protein concentrations were calculated using the BCA Protein Assay Kit (Beyotime, Shanghai, China). Equal amounts of protein (50 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked in 5% nonfat milk and then incubated overnight at 4 ℃ with the following primary antibodies: rabbit anti-Bax antibody (1∶2000; Cat. #50599-2-lg, Proteintech), rabbit anti-Bcl2 antibody (1∶2000; Cat. #4223T, Cell Signaling Technology, Massachusetts, USA, ), mouse anti-TLR4 antibody (1∶2000; Cat. #66350-1-1g, Proteintech), rabbit anti-MyD88 antibody(1∶2000; Cat. #23230-1-AP, Proteintech), rabbit anti-p65 antibody(1∶2000, Cat. #80979-1-RR, Proteintech), rabbit anti-p-p65 antibody(1∶2000; Cat. #82335-1-RR, Proteintech), and mouse β-actin monoclonal antibody (1∶5000; Cat. #66009-1-1g, Proteintech). βactin was used as a control. The next day, membranes were incubated with peroxidase-conjugated goat antirabbit or goat anti-mouse IgG for 1 h at room temperature and subjected to substrate development.

Total cell lysates (30 mg) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with goat anti-IkBa antibody (L-14, 1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-cleaved-PRAP antibody, (c-PARP, 1:500 dilution; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Bim antibody (1:1,000; Cell Signaling Technology), rabbit anti-Bcl2 antibody (1:500 dilution; Cell Signaling Technology), or mouse anti-b-actin antibody (cytosol marker, 1:1,000 dilution; Sigma-Aldrich). Signals were visualized using an ECL detection kit (Amersham Pharmacia Biotech, Little Chalfont, UK). Relative expression of proteins was quantified using ImageJ software.