Rabbit anti erk1 2

Proteins from harvested cells or skin tissues were extracted with RIPA lysis buffer containing protease inhibitors (ThermoFisher Scientific) and quantified using a BCA protein assay kit (Thermo Fisher Scientific, USA). Proteins were electrophoresed by SDS-PAGE and transferred to PVDF membranes which were blocked with 5 % nonfat milk. They were incubated with the corresponding primary antibodies at 4 °C overnight. Primary antibodies including rabbit anti-p65 (1:1,000, Cell Signaling Technology, USA), rabbit anti-phospho-p65 (1:1,000, Cell Signaling Technology, USA), rabbit anti-IκB (1:1,000, Cell Signaling Technology, USA), rabbit anti-phospho- IκB (1:1,000, Cell Signaling Technology, USA), rabbit anti-AKT (1:1,000, Cell Signaling Technology, USA), rabbit anti-phospho-AKT (1:1,000, Cell Signaling Technology, USA), rabbit anti-ERK1/2 (1:1,0000, Proteintech Group, China), rabbit anti-phospho-ERK1/2 (1:1,000, Proteintech Group, China), rabbit anti-TNF-α (1:1,000, Abcam, UK), rabbit anti-HSP90 (1:5,000, Proteintech Group, China) and rabbit anti-GAPDH (1:15000, Bioworld, China). The second day, PVDF membranes were incubated with secondary horseradish peroxidase-conjugated antibodies (1:10000 dilutions) at room temperature for 1 h. The protein expression was detected by the ChemiDocTM XRS + system (Bio-Rad).