Goat anti mouse igg ha1006

Nearly, 20-30 mg kidney tissue was homogenized in SDS-lysis buffer (#7722, CST) containing 42 mM DTT following the user manual. The same samples were loaded to the SDS-PAGE gels and run at 100 v for 1 h 40 min at RT in Tris-Glycin-SDS containing buffer. Blots were blocked with 5% non-fat dry milk powder in tris-buffer saline containing Tween-20 (TBST) for 30 min at RT. The blots were incubated in primary antibody overnight at 4 °C. After primary antibody (all the antibodies were shown in supplementary Data 4) incubation, blots were washed three times with TBST. Horseradish peroxidase-labeled goat anti-rabbit IgG (HA1001, 1:2000 dilution; HuaBio, Hangzhou, China) or goat anti-mouse IgG (HA1006, 1:2000 dilution; HuaBio, Hangzhou, China) were probed for 1 h at RT prepared in non-fat dry milk containing Tween-20. Finally, blots were washed with TBST for three minutes each 10 min at RT, and observed through Odyssey infrared imaging system. (Fluorescence Chemiluminescence Imaging System, Clinx Science, Shanghai, China) and quantified through utilizing ImageJ software (version 6.0; Wayne Rasband, National Institutes of Health, USA).

Proteins were extracted from kidneys or HK‐2 cells using RIPA lysis buffer (P0013B; Beyotime Biotechnology) containing 4% cocktail proteinase inhibitors. After centrifugation at 3000 g for 15 minutes at 4°C, the supernatant was collected, and protein concentration was determined using Pierce^™ BCA Protein Assay Kit (23225; Thermo Scientific). Bovine serum albumin was used as the standard. Equal amounts of protein lysate were loaded directly on 10%‐12% SDS‐PAGE and transferred onto PVDF membrane for protein blotting (162‐0177; Bio‐Rad). The membranes were blocked with 5% non‐fat dry milk (w/v) in TBS‐T for 1 hour at room temperature and then incubated with indicated primary antibodies overnight at 4°C. After being rinsed thrice with TBS‐T at 5‐minute intervals, the membranes were incubated with horseradish peroxidase‐labelled goat anti‐rabbit IgG (HA1001, 1:2000 dilution; HuaBio) or goat antimouse IgG (HA1006, 1:2000 dilution; HuaBio) for 1 hour. Immunoblots were visualized using the Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500; Millipore Corporation) with Bio‐Rad ChemiDoc MP. All immunoblot analysis data are from experiments performed in triplicate. Densitometry analysis was performed using ImageJ 6.0 software (National Institutes of Health).