Rabbit anti bcl2 antibodies

After treatment, hPDLSCs were washed with cold PBS and lysed in RIPA lysis buffer with the supplied with protease/phosphatase inhibitor cocktail. Protein concentrations were measured by BCA protein assay kits. Total protein (25 μg) was fractionated with SDS-PAGE gels and transferred onto PVDF membranes. The PVDF membranes were blocked with 5% skim milk and incubated with primary antibodies at 4°C overnight. Rabbit anti-Bcl2 antibodies (1: 1000), rabbit anti-Bax antibodies (1: 1000), rabbit anti-Phospho-cleaved caspase3 antibodies (1: 500), rabbit anti- caspase3 antibodies (1: 1000), rabbit anti-bmp2 antibodies (1: 1000), rabbit anti-Oct4 antibodies (1: 1000), rabbit anti-Sox2 antibodies (1: 1000), rabbit anti- Runx2 antibodies (1: 1000), rabbit anti-MyD88 antibodies (1: 1000), rabbit anti-NF-κB p65 antibodies (1: 500), rabbit anti-Phospho-NF-κB p65 antibodies (1: 1000), rabbit anti-TLR4 antibodies (1: 1000), and rabbit anti- GAPDH antibodies (1: 1000) were obtained from Cell Signaling Technology. After washing 3 times with PBS, appropriate secondary antibodies were applied for 2 h, and signals were analyzed using the Tanon-5200 Chemiluminescence Imager with enhanced chemiluminescence Western blotting substrate (Millipore, Billerica, MA).

Total proteins were obtained from PC12 cell lines by radioimmunoprecipitation assay buffer (RIPA) lysis buffer after being washed with cold phosphate-buffered saline (PBS). Total protein extract was fractionated with 10% SDS-PAGE and transferred onto PVDF membranes. Each membrane was incubated with specific primary antibodies overnight at 4°C after being blocked with 5% skim milk. Rabbit anti- NDRG4 antibodies (1: 1000), rabbit anti-p-eNOS antibodies (1: 1000), rabbit anti-eNOS antibodies (1: 1000), rabbit anti-iNOS antibodies (1: 1000), rabbit anti-Bcl2 antibodies (1: 1000), rabbit anti-Bax antibodies (1: 1000), rabbit anti-cleaved caspase-3 antibodies (1: 1000), and rabbit anti-caspase-3 antibodies (1: 1000) were acquired from Cell Signaling Technology (Danvers, MA, USA). After being washed with 1×TBST, 3 times, the PVDF membranes were reacted with appropriate horseradish peroxidase-conjugated secondary antibodies for 2 hours at room temperature. Further, the target proteins expression was analyzed by using chemiluminescence detection kit (Millipore, Billerica, MA, USA).

Lung tissues were homogenized to obtain protein samples with RIPA lysis buffer containing PMSF after washing with cold PBS. Then, the protein (25 μg) was separated by SDS-PAGE and transferred to PVDF membranes. The membranes were incubated overnight with appropriated primary antibody at 4°C after being blocked with 5% bovine serum albumin (BSA). Rabbit anti-ICAM-1 antibodies (1: 1000), rabbit anti-CXCR4 antibodies (1: 1000), rabbit anti-SDF-1 antibodies (1: 1000), rabbit anti-Bcl2 antibodies (1: 1000), rabbit anti-bax antibodies (1: 1000), rabbit anti-cleaved caspase3 antibodies (1: 500), rabbit anti-NLRP3 antibodies (1: 1000), rabbit anti-ASC antibodies (1: 1000), rabbit anti-pro-IL-1β antibodies (1: 1000), rabbit anti-IL-1β antibodies (1: 1000), rabbit anti-pro-caspase1 antibodies (1: 1000), and rabbit anti-caspase1 antibodies (1: 1000) were purchased from Cell Signaling Technology (Danvers, USA). Subsequently, PVDF membranes were incubated with the corresponding secondary antibodies and visualized using a Tanon-5200 Chemiluminescence Imager (Tanon, Shanghai, China) with enhanced chemiluminescence (ECL) method.