Anti p pak1

Serum creatinine (SCr) (lot#: C011-1-1), blood urea nitrogen (BUN) (#C013-1-1), serum uric acid (SUA) (#C012-1-1), triglyceride (TG) (#A110-1-1), total cholesterol (TC) (#A111-1-1), and urine protein (#C035-2-1) assay kits were from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Anti-α-smooth muscle actin (αSMA) antibody (#bs-0189R) was purchased from Beijing Biosynthesis Biotechnology Co.,Ltd. (Beijing, China). Anti-Rac1(#ab33186), anti-GTP-Rac1 (#ab33186), anti-p-PAK1 (#ab75599), anti-p38MAPK (#ab195049), anti-p-p38MAPK (#ab47363), anti-β-catenin (#ab32572), anti-Podocin (#ab50339), and anti-nephrin (#ab216341) antibodies were from Abcam (Cambridge, United Kingdom). Anti-β-actin (#66009-1-Ig), anti-fibroblast-specific protein-1 (FSP-1) (#16105-1-AP), anti-PAK1 (#21401-1-AP), anti-snail (#13099-1-AP), HRP goat anti-mouse IgG (#SA00001-1), and HRP goat anti-rabbit IgG (#SA00001-2) antibodies were from Proteintech (Chicago, United States). Trizol (#15596026) was from Thermo Scientific (MA, United States). A Reverse Transcription kit (#CW2569) was purchased from CWBio Co., Ltd. (Beijing, China). STZ (#WXBC8740V) was from Sigma-Aldrich Co. Ltd. (MO, United States). Metformin (#A181224) from Zhejian Yatai Pharmaceutical Co., Ltd. (Shaoxing, China).

Tissues stored in liquid nitrogen were ground into powder. HEC-1A and Ishikawa cells were collected after 48 h of transfection. RIPA lysis buffer (Solarbio, Beijing, China) was added into the tissues powder and cells to extract total protein. The total protein concentration was investigated by BCA kit (Beyotime, Shanghai, China). For protein separation, 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with 10 μL of total protein samples. The separated protein was transferred onto polyvinylidene fluoride (PVDF) membrane. Then 5% skimmed milk was added to block protein for 1 h. Primary antibodies was used to probe protein for 12 h at 4 °C. The primary antibodies used in this study were as follows: rabbit anti-PAK1, anti-p-PAK1 and anti-VEGFA (1:1000, Abcam, Cambridgeshire, UK), rabbit anti-CDC42 and anti-GAPDH (1:1000, Cell Signaling, Beverly, MA, USA). The PVDF membrane was then treated by goat anti-rabbit secondary antibody (1:5000, Solarbio, Beijing, China) for 1 h at room temperature. The protein blots were visualized using enhanced chemiluminescence (ECL) system (Pierce Biotechnology, Rockford, IL, USA) according to the instructions. Image J software (NIH, Bethesda, MD, USA) was used for the blots intensity detection. GAPDH was the internal control.