Ab196495

HK-2 cells and kidney tissues were homogenized in radioimmunoprecipitation assay buffer (Beyotime, Nanjing, China) containing protease inhibitors to lyse the cells to obtain total proteins. After collecting the proteins in each sample separately, the proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF membranes in an ice-water bath and incubated with primary antibody at 4 °C for 12 h after being closed with 5% skim milk for 1 h at room temperature. Primary antibody was used at the following dilutions: Bax (1:200, Cell Signaling, 2772); Bcl-2 (1:1000, Abcam, Ab196495); CHOP (1:1000, Cell Signaling, 2895); GRP78 (1: 1000, Abcam, Ab21685) and GAPDH (1:1000, Hangzhou Jiahe Biotechnology Co., AB-PR 001);JAK2(1:5000, Abcam, Ab108596);p-JAK2(1:1000, Abcam, Ab32101);STAT3(1:1000, Abcam, Ab68153);p-STAT3(1:1000, Abcam, Ab267373). The membranes were then washed three times with TBST for 20 min each, incubated for 2 h at room temperature with appropriate secondary antibodies, then washed three times with TBST for 10 min each, and finally the western blots were visualized using chemiluminescent HRP substrate (Millipore, Billerica, MA, USA). Data analysis was performed using Image J software (NIH, USA) to quantify protein levels.

Total protein was extracted with the use of radio‐Immunoprecipitation Assay lysate buffer (R0010; Beijing Solarbio Science & Technology Co., Ltd. Beijing, China). Protein concentration of each sample was determined by using a BCA kit (GBCBIO Technologies, Guangzhou, Guangdong, China). The protein was separated by polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane (Millipore) and sealed with 5% BSA at room temperature for 1 h. Primary rabbit antibodies (1:1000) from Abcam to cleaved‐Caspase3 (ab49822), Caspase3 (ab13847), B cell lymphoma‐2 (Bcl‐2; ab196495), Bcl‐2‐associated X (Bax; ab32503), FOS (ab190289) and p65 (ab16502) as well as primary rabbit antibody to p‐p65 (Ser536) (1:1000; 3033, Cell Signaling Technology) were added to the membrane and incubated overnight. The following day, the membrane was incubated with goat anti‐rabbit IgG (ab97051, 1:2000, Abcam) at room temperature. The immunocomplexes on the membrane were visualized using enhanced chemiluminescence reagent and imaged using Image Quant LAS 4000C gel imager (GE, General Electric Company, Schenectady, NY, USA). With rabbit anti‐β‐actin (1:3000; Abcam, ab8227) serving as an internal reference, the grey value of each band was analysed by gel image analysis software Image J.