P smad3

Kidney tissues were lysed with RIPA buffer to obtain total protein. After the protein concentration was determined by the BCA protein detection kit (Abcam, UK), the total protein was performed with sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and electrotransfer. Then, the membrane is sealed with 5% completely skimmed milk powder, add the diluted primary antibody solution (NF-κB p65, 6535-1-Ig, Proteintech), (NLRP3, DF7438, Affinity), (caspase-1, AF5418, Affinity), (α-SMA, AF1032, Affinity), (smad3, AF6362, Affinity), (p-smad3, AP0727, Abclonal) and incubate the membrane at 4 °C for 12 h. Next, the membrane was washed with PBST (every 15 min) four times, then incubated with the diluted secondary antibody at room temperature for 2 h to enhance the chemiluminescence detection system immune signal. β-actin as a reference and Image J software was used for optical density analysis.

Cell lysates were first collected and extracted in RIPA buffer (Millipore, Burlington, MA, USA) 24, 48, and 72 h post-transfection with either siPDIA-4 or scrambled controls. Equal amounts of protein were loaded onto SDS-polyacrylamide gels and transferred to the PVDF membranes (Thermo Fisher, Madison, WI, USA). Membranes were blocked for 1 h in 1X TBS with 3% BSA and incubated overnight at 4 °C with primary antibodies. The primary antibodies used in this study include p21 (Cell Signaling #2947S), Cleaved Caspase-3 (Cell Signaling #9664S), LC3 I/II (Cell Signaling #4600IAP), GAPDH (Cell Signaling #5174S), p-SMAD3 (ABclonal #51451), SMAD3 (ABclonal #28379), Βeta Actin (Cell Signaling #4970S), Βeta Tubulin (Proteintech Cat#66240-1-Ig), Bax (Cell Signaling #2772S), Bcl-2 (Cell Signaling #3498S), CD31 (ABclonal Cat#A4900), α smooth muscle actin (ABclonal Cat#A17910), N-Cadherin (ABclonal Cat#A19083), PDIA-4 (Cell Signaling #5033S), and P53 (Santa Cruz #sc-126). Proteins were then incubated with goat anti-rabbit secondary antibody (Enzo ADI-SAB-300-J) for 2 h at room temperature. Bands were visualized with the ECL substrates using a chemiluminescence channel and 700 channels in the LiCor Fc Odyssey system. The bands were quantified using LiCor Fc Odyssey inbuilt software version 5.2.

SKI was purchased from Xi’an Century Shenkang Pharmaceutical Industry Co., Ltd. (Xi’an, China, 202102103). TGF-β1 (Santa Cruz, sc-130348), E-Cad (CST, #3195), P-Smad3 (Abclonal, A19115), P-Smad2/3 (Abclonal, A19115), Smurf2 (Santa Cruz, sc-518164), α-SMA (CST, #19245), collagen I (Abclonal, A5786), Smad7 (Santa Cruz, sc-365846), Smad2/3 (Santa Cruz, sc-133098), ubiquitin (Santa Cruz, sc-8017), TβR-I (Santa Cruz, sc-101574), TβR-II (Santa Cruz, sc-1700), Smad3 (Santa Cruz, sc-101154), serum creatinine (Scr), and blood urea nitrogen (BUN) assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Real-Time PCR Easy™- SYBR Green I (FOREGENE, QP-01014).