Hrp β actin antibody ac 15

The renal proteins were homogenized with a lysis buffer containing: 50 mM HEPES pH 7.4, 250 mM NaCl, 5 mM EDTA, 0.1% NP‐40 and complete protease inhibitor (Roche, cat. no. 11697498001). The proteins concentration was assessed by Lowry protein assay (Bio‐Rad, Cat. No. 5000113 and 5000114). Renal protein levels were detected by Western blot, tissue proteins (20 μg) were electrophoresed in a denaturing 8.5% acrylamide gel with SDS. The membranes were incubated with the primary antibody IL‐6 (Santa Cruz, cat. no. sc57315, 1:1000), BiP‐1 (Cell Signaling, cat. no. 3177, 1:1000), CHOP (Cell Signaling, cat. no. 2895, 1:5000), FOXO3 (Santa Cruz, cat. no. sc11351, 1:5000), Mitofusin‐1+Mitofusin‐2 (Abcam, cat. no. ab57602, 1:1000), Drp1 (Santa Cruz, cat. no. sc‐271583, 1:1000) and HRP β‐Actin antibody [AC‐15] (Abcam, cat. no. ab49900, 1:1,000,000) overnight at 4℃. Three of 10‐min washes were performed with TBS‐1x Tween, and then incubated with a secondary antibody coupled to HRP, anti‐rabbit or anti‐mouse IgG (Santa Cruz, cat. no. sc‐2031 or sc‐2004, respectively 1:5000). Tissue proteins assessed by Western blot were normalized by β‐actin detection.

The renal cortex proteins were homogenized with a lysis buffer containing: 50 mM HEPES pH 7.4, 250 mM NaCl, 5 mM EDTA, 0.1% NP-40 and complete protease inhibitor (Roche, Cat. No.. 11,697,498,001). The proteins concentration was assessed by Lowry protein assay (Bio-Rad, Cat. No. 5000113 and 5,000,114). Renal cortex protein levels were detected by Western blot, tissue proteins (20–40 µg) were electrophoresed in a denaturing 8.5% acrylamide gel with SDS. The samples were prepared with loading buffer in a 1:1 ratio with a final volume of 20 μL. The membranes were incubated with the primary antibody TGFβ (Thermo Fisher, Cat. No. MA5-15,065, 1:1000), HIF-1alpha (Abcam, Cat. No.. ab2185, 1:5000) VEGFA (Invitrogen, MA1-16,629, 1:5000), IL-6 (Abcam, Cat. No. ab9324, 1:1000), and HRP β-Actin antibody [AC-15] (Abcam, Cat. No. ab49900, 1:1,000,000) overnight at 4 °C. Three 10-min washes were performed with TBS-1 × Tween and then incubated with the secondary antibody coupled to HRP, anti-rabbit or anti-mouse IgG (Santa Cruz, Cat. No. sc-2031 or sc-2004, respectively 1: 5000). Tissue proteins assessed by Western blot were normalized by β-Actin detection.