2.8. Western Blotting

Total protein was isolated with the RIPA lysis buffer (Solarbio, Beijing, China) and added with 1% protease inhibitor (Solarbio) from cells. Subsequently, same amount of proteins (about 20 μg) from each group were separated by 10% SDS-polyacrylamide gelsis and transferred onto the polyvinylidene difluoride membranes (PVDF; Millipore, Billerica, MA, USA). After that, the membranes were blocked with 5% non-fat milk at room temperature for 60 min to prevent the nonspecific bindings, followed by primary antibody incubation at 4 °C for overnight, including anti-β-actin antibody (cat no. ab8226, Abcam, MA, USA; 1 : 5000 dilution), anti-LIMS2 antibody (cat no. ab272666, Abcam; 1: 2000 dilution), anti-p-ERK (cat no. 4370, CST; 1: 2000 dilution), anti-ERK (cat no. 4695, CST; 1: 2000 dilution), anti-p-P38 (cat no. 4511, CST; 1: 1000 dilution), anti-P38 (cat no. 8690, CST; 1: 1000 dilution), anti-p-JNK (cat no. 9251, CST; 1: 1000 dilution), and anti-JNK (cat no. 9252, CST; 1: 1000 dilution) antibodies. After that, the membranes were probed with HRP-conjugated secondary antibodies at room temperature for 1 hour. ProfiBlot-48 (Tecan, Switzerland) was applied to evaluate protein signaling following immersing in ECL reagent (Millipore, USA). ImageJ software was used for protein quantification.