Erk1 2

These two methods have been depicted in detail in our previous study [28 (link)]. The primer sequences are summarized in Table 1. The primary antibodies in this study included CGRP (ab283568, 1 : 1000, Abcam, USA), CALCRL (861587, Zen Bio, Chengdu, China), RAMP1(385544, Zen Bio, Chengdu, China), Bax (200958, 1 : 1000, Zen Bio, Chengdu, China), Bcl2 (381702, 1 : 1000, Zen Bio, Chengdu, China), Cleaved-caspase3, iNOS (1 : 1000, Zen Bio, Chengdu, China), COX-2 (1 : 1000, Zen Bio, Chengdu, China), MMP3 (384995, 1 : 1000, Zen Bio, Chengdu, China), type I collagen (1 : 1000, ab34710, Abcam), type II collagen (1 : 1000, Zen Bio, Chengdu, China), aggrecan (ab36861, 1 μg/mL, Abcam, USA), NF-κB p65 (D14E12, #8242, Cell Signaling Technology, Inc., three Trask Lane Danvers, United States), p-p65 (Ser536; #3033, Cell Signaling Technology, Inc., three Trask Lane Danvers, United States), Ik-Ba (380682, 35 kDa; Zen Bio, Chengdu, China, 1 : 1,000), p-Ik-Ba (340776, 35 kDa; Zen Bio, Chengdu, China, 1 : 1,000), ERK1/2 (201245-4A4, 42/44 kDa; Zen Bio, Chengdu, China, 1 : 1,000), p-ERK1/2 (301245, 42/44 kDa; Zen Bio, Chengdu, China, 1 : 1,000), p38 (200782, 43 kDa; Zen Bio, Chengdu, China, 1 : 500), p-p38 (310069, 43 kDa; Zen Bio, Chengdu, China, 1 : 1,000), JNK (381100, 46/54 kDa; Zen Bio, Chengdu, China, 1 : 1,000), and p-JNK (380556, 46/54 kDa; Zen Bio, Chengdu, China, 1 : 1,000).

The proteins of cells and kidney samples were separated, and then isolated through SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skimmed milk powder for 2 h and incubated with the primary antibodies, which including KIM-1 (1:3000; Novus, Colorado, USA), cleaved Caspase-3 (1:1000; Cell Signaling, Danvers, MA, USA), Bcl-2 (1:1000; Bioworld Technology, Nanjing, China), ERK1/2 (1:2000; ZEN-BIOSCIENCE, Chengdu, China), Phospho-ERK1/2 (1:1000; ZEN-BIOSCIENCE, Chengdu, China), Akt (1:1000; ZEN-BIOSCIENCE, Chengdu, China), Phospho-Akt (1:1000; ZEN-BIOSCIENCE, Chengdu, China), FGF2 (Bioworld, Nanjing, China), Phospho-FGFR1 (1:2000; Bioss, Beijing, China), GAPDH (1:5000; Bioworld, Nanjing, China) and β-actin (1:5000; Bimake, Houston, USA) overnight at 4°C. After cleaning with TBST, the membranes were incubated with Goat Anti-rabbit HRP antibody (1:5000; Bioss, Beijing, China) at room temperature for 1 h and the signals were detected with the Omin-ECL ultra-sensitive chemiluminescence detection kit (EpiZyme, Shanghai, China). The results were analyzed by ImageJ program.

The harvested cells were lysed by ultrasonication. The proteins were transferred to PVDF membranes (Millipore Corporation, Billerica, MA, USA) after gel electrophoresis (SDS-PAGE). The membranes were incubated at room temperature with 5% defatted milk powder for 1 h and then probed with the indicated primary antibodies: cleaved caspase-3, cleaved caspase-9, Bax, Bcl-2, LC3, beclin-1, P62, PI3K, p-PI3K, AKT, p-AKT, mTOR, p-mTOR, ERK1/2, p-ERK1/2, JNK1/2, p-JNK1/2, P38, p-P38 (all from Zen Bioscience, Chengdu, China) at 4 ℃ overnight. Next, the following secondary antibody was employed and incubated 2 h at room temperature. The signal was developed by the ECL detection system, and the relative expression of proteins was analyzed using the Quantity One software.