Wl02400

The isolated spinal cords were homogenated with RIPA buffer (Beyotime Biotechnology, Shanghai, China) containing 1% protease inhibitor (Beyotime Biotechnology, Shanghai, China) for 30 minutes on ice. The homogenates were centrifuged at 12000 g for 25 minutes at 4°C and supernatants were collected. NanoDrop 2000C (Thermo Scienti c, USA) was then used for protein concentrations determination. The homogenates and loading buffer 5× (Beyotime Biotechnology, Shanghai, China) were mixed at a ratio of 4: 1, denatured in the metal bath (100°C, 8~10 mins), cooled to room temperature and loaded. Protein samples were subjected to 12.5% SDS-PAGE (Epizyme Biotech, Shanghai, China) and then transferred to polyvinylidene uoride (PVDF) membranes. Membranes were blocked in protein free rapid blocking buffer (Epizyme Biotech, Shanghai, China) for 15 minutes at room temperature. After blocking, membranes were incubated with the primary antibodies against ATF3 (DF6660, A nity Biosciences), XBP1 (WL00708, WanleiBio), HMOX1 (WL02400, WanleiBio), DDIT3 (GADD153, Proteintech), CHAC1 (DF9353, A nity Biosciences) and GAPDH (AF7021, A nity Biosciences) overnight at 4°C. Membranes were incubated with the secondary antibody at room temperature for 1.5 h. Protein bands were captured using an ECL chemiluminescence system (Epizyme Biotech, Shanghai, China).