Rabbit anti mouse gapdh

Transfected PDSCs were cultured in OM under hypoxia for 24 or 48 h after transfection. For the rescue experiment, PI3K specific activator 740-YP 30 μg/ml (MedChemExpress, NJ) and AKT activator SC79 5 μg/ml (Sangon Biotech, Shanghai, China) were utilized. The total proteins were extracted using RIPA (Thermo Fischer Scientific, Waltham, MA). The proteins were denatured at 100°C for 10 min, separated using SDS-PAGE, and then transferred to the PVDF membrane (Millipore, Billerica, MA). Subsequently, the membranes were blocked with non-fat milk, incubated with primary antibody HIF-1α (1:2000), POSTN (1:3000), rabbit anti-mouse PI3K (1:1000, Cell Signaling, Danvers, MA), rabbit anti-mouse p-PI3K (1:1000, Cell Signaling, Danvers, MA), rabbit anti-mouse AKT (1:3000, Proteintech, Chicago, IL), rabbit anti-mouse pAKT (1:1000, Cell Signaling, Danvers, MA), and rabbit anti-mouse GAPDH (1:10000, Proteintech, Chicago, IL) at 4°C; washed with TBST; and incubated with secondary antibody for 1 h. The membranes were visualized using a chemiluminescence imaging system (Tanon, Shanghai, China) and semi-quantified using Image J.

Antibodies used in the study included rabbit anti-mouse caspase-1 p10 (sc-514) and anti-mouse ASC (sc-22514-R) (Santa Cruz, CA, United States); rabbit anti-mouse NLRP3 (Abcam, Cambridge, MA, United States); rabbit anti-mouse LC3B (MBL, Japan); and rabbit anti-mouse β-actin (Beyotime, Wuhan, Hubei, China); rabbit anti-rat TLR4 (Proteintech, Chicago, IL, United States); rabbit anti-mouse TRIF (Abcam, Cambridge, MA, United States); rabbit anti-mouse GAPDH (Proteintech, Chicago, IL, United States); goat anti-rabbit secondary antibody and goat anti-mouse secondary antibody (ZSGB Biotechnology, Beijing, China). Lipopolysaccharides (LPS, E. coli L2630) and 3-MA (M9281) were from Sigma-Aldrich (St. Louis, MO, United States), and rapamycin was from Beyotime Biotechnology (Wuhan, Hubei, China). Caspase-1 inhibitor Z-YVAD-FMK was purchased from Biovision (San Francisco, CA, United States). The ELISA kit for mouse IL-1β (88-7013) was purchased from eBioscience Technology (San Diego, CA, United States). Reagents and apparatus used in immunoblotting assays were obtained from Bio-Rad (Hercules, CA, United States).