Western Blot Analysis of Signaling Pathways

Cells were cultured and treated as previously described in a 6-well plate. Cell protein was extracted using Total Protein Extraction Kits, and quantified using BCA Protein Quantification Kits (Vazyme, China). For each sample, 20 μg protein was separated in 12% SDS-PAGEs. The protein bands were transferred to a polyvinylidene fluoride (PVDF) membrane and then blocked in Tris-buffered saline containing 0.05% Tween 20 and 5% bovine serum albumin for 2 h. The PVDF membranes were incubated at 4°C overnight with specific primary antibodies (1:1000 dilution in TBST) against p65, p-p65, IκBα, p-IκBα, p38, p-p38, JNK, p-JNK, ERK1/2, and p-ERK1/2. On the next day, the PVDF membranes were washed with TBST 5 times for 8 min each, and incubated with Rabbit Anti-Goat IgG/HRP (Bioss, China) antibody in 1:1000 dilution in TBST for 1 h at room temperature. After washing with TBST as described above, the protein bands were visualized with ECL western blotting detection reagent (Bioground, China). The experiments were repeated three times. β-actin protein was used as an endogenous control as in the previous report in MAC-T cells [20 (link)]. Band intensity normalized to β-actin was evaluated using Image Lab software (version 5.2.1, Bio-Rad).