P jak2

EC-109, EC-109 Vector and EC-109 shPITPNM3 cells were treated with or without 40 ng/mL rCCL18 in the presence or absence of STAT3 inhibitor S3I-201 (MCE, China). The total cell lysates were prepared using protein lysis buffer. Then protein was fractionated by 10% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membrane (Merck Millipore, IPVH00010, USA), and then blocked with 5% defatted milk dissolved in PBS (pH7.2) containing 0.1% Tween 20 for 2 h at room temperature. The PVDF membranes were incubated with the following primary human antibodies at 4 °C overnight: JAK2 (Abcam, AB32101, UK), P-JAK2 (Abcam, AB108596, UK), STAT3 (CST, 4904S, USA), P-STAT3 (CST, 9145 T, USA). The reference antibody was β-actin (Servicebio, China). Thereafter, the PVDF membranes were incubated with secondary antibody (Sangon Biotech, D110011-0100, China), the blots were visualized using ECL system (Azure C600, USA).

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 of the TSPCs was extracted in cold RIPA lysis buffer (Keygen biotech), and the protein concentration was measured using BCA protein assay kit (Thermo Scientific). A total of 30 μg of protein was electrophoresed on SDS-PAGE. After electrotransfer onto PVDF membrane (Millipore), the membranes were blocked with PBST containing 5% non-fat dry milk for 1 h at room temperature. The membranes were then incubated with primary antibody against JAK2 (Proteintech), p-JAK2 (Abcam), STAT3 (Proteintech), p-STAT3 (Abcam), p16^INK4A (Beyotime Biotechnology), cyclin D1 (Bioworld), cyclin B1 (Proteintech), and GAPDH (Proteintech) at 4°C overnight. After washing with PBST, the membranes were incubated with secondary antibody and immunoreactive bands were visualized with ECL reagents (Keygen biotech).

Total protein was extracted by lysing cells in RIPA buffer containing protease inhibitor. Protein samples were separated by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred onto polyvinylidenefluoride (PVDF) membranes. After blocking with 5 % non-fat milk or 3 % BSA in TBS-T, membranes were incubated with the primary antibody. The following antibodies were used: SH2B1 (1:1000,Abcam, USA), JAK2 (1:600, Abcam, USA), p-JAK2 (1:2000, Abcam, USA), p-Rac1 (1:500, Abcam, USA), Anti-cAMP Protein Kinase Catalytic subunit (1:60000,Abcam,USA),MMP2 (1:2000,Abcam.,USA), MMP9 (1:1000, Abcam,USA), GAPDH (1:10000, Abcam, USA) and goat-anti-rabbit IgG conjugated to horseradish peroxidase (HRP) (1:5000, Santa Cruz, USA),which was used as the secondary antibody. Cells were seeded on 10 cm cell culture plates, grown to 80 % confluences, and serum starved overnight. Target signals were quantified by BandScan software (Bio-Rad, Hercules, CA) and defined as the ratio of target protein relative to GAPDH.