Phospho akt1

Quiescent mesangial cells were stimulated with PDGF-AB in the presence or absence of AXL inhibitor R428 for 15 minutes. Cells were then washed and lysed with M-PER lysis buffer supplemented with protease and phosphatase inhibitors and the lysates were processed as described. Immunoblotting used antibodies specific for PDGFR-β (R&D Systems), phospho-PDGFR-β (Cell Signaling Technology), ERK1/2 (Cell Signaling Technology), phospho-ERK1/2 (R&D Systems), AKT1 (Santa Cruz Technology), phospho-AKT1 (Santa Cruz Technology), AXL, and phospho-AXL (R&D Systems), and the blots were developed as described.

Immunohistochemistry was performed using a Streptavidin Biotin Complex immunohistochemistry kit (Boster Bioengineering Co., Wuhan, China). The paraffin sections were dewaxed and antigen was repaired and sealed with goat serum. Then the sections were incubated overnight at 4°C with primary antibodies [phospho-PI3K (p-PI3K; 1:2000; mouse; Cat# sc-12929; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), phospho-Akt 1/2/3 (1:2000; mouse; Cat# sc-101629; Santa Cruz Biotechnology Inc.), and vascular endothelial growth factor (1:2000; mouse; Cat# sc-365578; Santa Cruz Biotechnology Inc.)], followed by incubation at 37°C for 30 minutes with horseradish peroxidase-labeled goat anti-mouse IgG(H+L) (1:2000; Cat# ZB-5305; Zhongshan Jinqiao Biotechnology Co. Ltd., Beijing, China) on the next day. After 3,3’-diaminobenzidine staining and hematoxylin staining, photographs were taken under the light microscope.