D085075

Based on a previously reported protocol [1 (link)], the dorsal neurovascular bundle and buck fascia were removed from the TA and urethra. The tissues and fibroblasts in each group were lysed in RIPA buffer (R0010, Solarbio, China). Twelve per cent SDS-PAGE was used to separate proteins (30 μg), and the separated proteins were transferred to PVDF membranes (EMD Millipore). Appropriate primary antibodies diluted with 5% BSA were incubated with the membranes overnight at 4 °C. The primary antibodies consisted of collagen 3 (1:800, AF5457, Affinity, China), Smad7 (1: 800, AF5147, Affinity, China), elastase-2B (1: 800, orb471854, Biorbyt, China), osteopontin (1: 800, AF0227, Affinity, China) and GAPDH (1:1000, BA2913, Boster, China). After 3 washes with TBS-0.01% Tween 20, the HRP-Affinipure goat anti-rabbit IgG (H + L) (1:500, BM3894, Boster, China) was incultured with the membranes for 2 h at 25 °C. After washing, the signals were visualized using enhanced chemiluminescence reagent (D085075, Bio-Rad, China).

The damaged hippocampal region tissues were separated with RIPA buffer (Beyotime, China) on ice and homogenized to extract protein. Forty μg proteins were separated with 12% SDS-PAGE (Bio-Rad, China) and transferred to PVDF membranes (EMD Millipore). After blocking with 5% milk for 1.5 h, the membranes were incubated with appropriate primary antibodies diluted with 5% BSA for overnight at 4 °C. The primary antibodies consisted of phospho-PPARγ (ser273) (1:1000, bs-4888R, Bioss, China), LC3BI/II (1:1000, 4108, Cell signaling technology, China), phospho-NF-κB p65 (1:800, bs-230303R, Bioss, China), GAPDH (1:1000, bs-0755R, Bioss, China). After washing with TBS-0.01% Tween 20 for 3 times (10 min/wash), the secondary antibody Goat Anti-rabbit lgG/HRP (1:1000, bs-0295G-HRP, Bioss, China) was cultured with the membranes for 2 h at 25 °C. After washing, the signals were visualized using enhanced chemiluminescence reagent (D085075, Bio-Rad, China).