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. 2020 Jan 27;318(3):F647–F659. doi: 10.1152/ajprenal.00288.2019

Fig. 6.

Fig. 6.

18E1 monoclonal antibody (mAb) reduces epidermal growth factor (EGF) receptor (EGFR) and ERK1/2 phosphorylation in podocytes. A: cultured mouse podocytes were treated with EGF (20 ng/mL) for 15 min with or without 10 µg/mL 18E1 mAb or control IgG. Phosphorylation of EGFR (immunoprecipitated), ERK1/2, and Akt was assessed by immunoblot analysis using phosphospecific antibodies against EGFR (Tyr1173), ERK1/2 (Thr202/Tyr204), and Akt (Ser473). The antibodies were stripped, and membranes were reblotted with antibodies to total EGFR, ERK1/2, or Akt. The ratios of phosphorylated protein to total protein are shown. The ratio in the unstimulated condition is expressed as 1.0. B: specificity of the 18E1 mAb effects was assessed by CD148 knockdown. CD148 knockdown was carried out using lentivirus encoding mouse CD148-targeting shRNA (shRNA #1 and shRNA #2). Lentivirus encoding scrambled shRNA was used as a control. CD148 knockdown was confirmed by immunoblot analysis (top). Effects of 18E1 mAb in EGFR and ERK1/2 phosphorylation were assessed in control (a) and CD148 knocked down (b and c) cells as in A. Note that CD148 knockdown largely diminished the 18E1 mAb suppression of EGFR and ERK1/2 phosphorylation. C: mouse podocytes were cultured in normal glucose (NG; 5.6 mM) and high glucose (HG; 25 mM) conditions for 48 h with either 18E1 or control IgG (10 µg/mL). Phosphorylation of EGFR and ERK1/2 was assessed as in A. Note that 18E1 mAb reduced HG-induced EGFR and ERK1/2 phosphorylation compared with control IgG. Representative data of five independent experiments are shown in A−C. D: immunostaining of EGFR and ERK1/2 phosphorylation in 18E1 mAb- or control IgG-treated diabetic mouse glomeruli. The glomeruli in nondiabetic control mouse treated with control IgG are also shown. Immunofluorescence staining of podocin (red) was used to identify podocytes. Arrows indicate podocytes. Representative data of eight mice are shown. Note that treatment with 18E1 mAb reduced EGFR and ERK1/2 phosphorylation in podocytes in diabetic mice. Scale bar = 25 µm.