Enhance chemiluminescence

Total cellular protein was extracted with the total protein extraction kit (KeyGen Biotech, Nanjing, China). Protein concentration was determined using a BCA protein assay kit (Key-Gen Biotech). Equal amounts of protein were analyzed using 10%SDS–PAGE, and transferred to a PVDF membrane. Anti- CatK and anti-GAPDH antibodies were diluted at 1∶500 and 1∶4000 respectively. Primary antibodies against P- JNK, JNK, P-p38, p38, P-Jun, Jun, P-MAPKAPK-2 and Fos were diluted at 1∶1000, and incubated with membranes overnight at 4°C. Following subsequent incubation with a secondary antibody, blots were visualized with enhance chemiluminescence (Millipore, Billerica, MA, USA). Bands on X-ray film were scanned with GS-800 Calibrated Densitometer (Bio-rad, USA). The intensity of bands was quantified with Quantity one software. All values were normalized to the corresponding GAPDH.

Total cellular protein was extracted with the protein extraction kit (KeyGen Biotech, Nanjing, China). The modified Lowry protein assay kit (Pierce, Rockford, IL, United States) was used to quantify protein. Equal amounts of protein were analyzed using 10% SDS–PAGE, and transferred to a PVDF membrane. PVDF membranes were incubated with primary rabbit anti-LC3B (CST, 3868S,1:1200), primary mouse anti-cathepsin B (ab58802,1:1000), rabbit anti-P53 (ab131442,1:1000), rabbit anti-p21Cip1p (CST,2947S,1:1000), rabbit anti-p16INKa (CST,80772S,1:1000), rabbit anti-P62 (CST,16177S,1:1000), and anti-GAPDH (ab181602,1:2000) incubated overnight at 4°C. Following subsequent incubation with anti-rabbit HRP-conjugated secondary antibody (Affinity, S0001, 1:2000) or anti-mouse HRP-conjugated secondary antibody (Affinity, S0002, 1:2000), blots were visualized with enhance chemiluminescence (Millipore, Billerica, MA, United States). Bands on X-ray film were scanned with GS-800 Calibrated Densitometer (Bio-Rad, Hercules, CA, United States). The intensity of bands was quantified with Quantity one software. All values were normalized to the corresponding GAPDH.