Hrp labeled donkey anti goat igg

The spinal cord tissues (L3-L4) were homogenized in a lysis buffer containing Protease Inhibitor Cocktail (p8340, Sigma-Aldrich) [19 (link)]. The protein concentration was confirmed with BCA protein concentration determination assay kit (P0010, Beyotime, Shanghai, China). The protein samples were separated by SDS-PAGE gels and transferred onto polyvinylidene fluoride membrane (IPVH00010, Millipore). The membrane was sealed with 5% skim milk for 2 h at room temperature, subsequently incubated at 4°C overnight with the primary antibodies: Mtf1 (1 : 500; Santa Cruz Biotechnology, CA, USA), p-ERK1/2 (1 : 5000; Sigma), ERK1/2 (1 : 1000; Santa Cruz Biotechnology), GFAP (1 : 1000; Cell Signaling Technology, Danvers, MA, USA) and β-actin (1 : 2000; Santa Cruz Biotechnology). The membrane was washed for 5 min/time 3 times at room temperature, incubated for 2 h in the corresponding secondary antibody: HRP-labeled goat anti-mouse IgG (1 : 1000; Beyotime), HRP-labeled goat anti-rabbit IgG (1 : 1000; Beyotime) and HRP-labeled donkey anti-goat IgG (1 : 1000; Beyotime) at room temperature. The membrane was then washed for 5 min/time 6 times, the immune complexes were detected by ECL chemiluminescent assay kit (Biosharp, Guangzhou, China). Signal intensity of band analyses was conducted with ImageJ software (Alliance Q9).

Equal amounts of protein (20–30 μg) were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane (PVDF, Millipore, Billerica, MA, USA). The membrane was blocked for 1 h with 5% fat-free dried milk at RT, then incubated with primary antibodies (Santa Cruz Biotechnology) against A3AR (1:800 dilution), NF-κB p65 (1:600 dilution), IκB-α (1:400 dilution), p-IκB-α (1:400 dilution), Tubulin (1:1,000 dilution), or β-actin (1:1,000 dilution) at 4°C overnight. Membranes were washed three times with Tris-buffered saline with Tween-20 (TBS-T) and incubated with the corresponding secondary antibodies (HRP-labeled Goat Anti-mouse IgG, HRP-labeled Goat Anti-rabbit IgG, HRP-labeled Donkey Anti-goat IgG; 1:1,000; Beyotime, China) for 1 h at room temperature. Signals were detected with an electrochemiluminescence (ECL) detection reagent (Beyotime, China). The images were obtained on Kodak film and quantified by Quantity One software (Bio-Rad, Hercules, CA, USA).