Hrp labeled anti mouse igg antibody

2-Octen-1-ylsuccinic anhydride, RPMI 1640 medium, fetal bovine serum (FBS), penicillin, streptomycin, polymyxin B sulfate salt, and LPS from Escherichia coli 026/B6 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α and IL-6 were purchased from R&D Systems (Minneapolis, MN, USA) and Biolegend (San Diego, CA, USA), respectively. Goat anti-actin antibody and anti-goat IgG antibody labeled with horseradish peroxidase (HRP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-IκBα antibody, HRP-labeled anti-mouse IgG antibody, HRP-labeled anti-rabbit IgG antibody, and rabbit antibodies against histone H3, NF-κB p65, ERK1/2, phosphorylated ERK1/2, JNK, phosphorylated JNK, p38 MAPK, and phosphorylated p38 MAPK were purchased from Cell Signaling Technology (Danvers, MA, USA). TAK-242 was purchased from Chemscene (Monmouth Junction, NJ, USA).

The cells and CSps cultured on the PS, ULA, and OPC substrates were lysed in RIPA buffer (Solarbio) for protein extraction. The protein concentrations of the samples were adjusted by bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific) according to the manufacturer’s protocol. Forty micrograms of protein was loaded into each lane of a 10% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (Millipore) gel and transferred to polyvinylidene fluoride membranes (Millipore). After being blocked with 5% bovine serum albumin in Tris buffered saline Tween (Biosharp) at room temperature for 1 h, the membranes were incubated with the primary monoclonal antibodies against caspase-1 (1:500, ab1872, Abcam) and β-actin (1:1000, ab8226, Abcam) in TBST overnight at 4 °C, after which HRP-labeled Anti-Rabbit IgG antibody (1:2000, 7074, Cell Signaling Technology) and HRP-labeled Anti-mouse IgG antibody (1:2000, 7076, Cell Signaling Technology) was added and incubated for another 1 h. The results were visualized via enzyme-linked chemiluminescence by an ELC kit (Thermo Fisher Scientific). β-Actin was used as an internal control.