Anti β arrestin 1

Western Blotting analysis was performed as described previously 32 (link). Total cell extracts were lysed in cell lysis buffer (Beyotime, P0013), and the lysates were centrifuged at 12,000 rpm for 10 min at 4°C. Samples containing 30 µg protein were loaded on SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked with 5% nonfat milk or BSA and probed with primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Blots were developed with lumi-light Western blotting substrate detection reagents (Roche, 12015200001). Nuclear- and cytoplasmic-enriched fractions were prepared using Nuclear-Cytosol Extraction Kit (Applygene, P1200), according to manufacturer's instructions. Western Blotting analysis was performed using the anti-p27 (Cell Signaling Technology, 3698S), anti-CREB (Cell Signaling Technology, 9197L), anti-phospho-CREB (Ser133) (Cell Signaling Technology, 9198L), anti-S-phase kinase-associated protein 2 (Skp2) (Cell Signaling Technology, 4313S), anti-CDK2 (Cell Signaling Technology, 2546S), anti-Cyclin E1 (Cell Signaling Technology, 4129S), PARP (Cell Signaling Technology, 9532S), anti-GAPDH (Cell Signaling Technology, 2118L), anti-β-arrestin 1 (Abcam, ab32099), anti-β-arrestin 2 (Abcam, ab54790), anti-tubulin (Beyotime, AT819), anti-cyclin D3 (Beyotime, AC856), anti-p21 (Beyotime, AP021).

Normal rat kidney tubule epithelial cells (NRK‐52E cells), and HEK‐293 cells were cultured as described. Before the experiment, cells were starved overnight in a medium containing 0.5% FBS and then treated with drugs. AT1R‐β‐ARR biased agonist SII ([1‐sar, 4, 8‐ile]‐angiotensin II) was purchased from Jill biochemical and dissolved in ddH₂0 at a stock solution of 10 mM. Angiotensin II human was purchased from Sigma‐Aldrich (cat# 4474‐91‐3). PD98059 was purchased from Selleck (cat# 167869–21‐8).
The antibodies used for Western blot analysis included (a) anti‐ LOX (no. 31238), anti‐β‐arrestin1 (no. 32099), anti‐elastin (no. 21610), anti‐Col1 (no. 34710), and anti‐GAPDH (no. 82451), purchased from Abcam; (b) anti‐ ERK1/2 (no. 4695), and anti‐p‐ERK1/2 (no. 4370), purchased from Cell Signaling Technology; and (c) anti ‐STAT3 (sc‐482), anti‐STAT3‐pS727 (no. sc‐800R) and anti‐STAT3‐pY705 (sc‐7993) were purchased from Santa Cruz Biotechnology Inc. The secondary antibodies, including donkey anti‐rabbit IgG–horseradish peroxidase (sc‐2313), and goat anti‐mouse IgG–horseradish peroxidase (sc‐2005) were purchased from Santa Cruz Biotechnology Inc.