Beta actin

The rats were euthanized and decapitated, and their ischemic ipsilateral cortices were rapidly dissected on ice and stored at −80 °C until needed. The samples were then taken from the freezer and sonicated in RIPA buffer (Beyotime Institute of Biotechnology, China) containing phosphatase and protease inhibitor cocktails. A BCA Kit (Beyotime Biotechnology) was then used to determine the protein concentrations. Equal amounts of sample were separated by SDS-PAGE and transferred onto PVDF membranes, which were blocked in 5% non-fat dry milk at room temperature for 1 h. The membranes were then incubated overnight at 4 °C with primary antibodies against VEGF (1:1000 Bioworld Technology, USA), Ang-1 (1:1000 Bioworld Technology, USA), Shh (1:250 Life Technology, USA), Patched-1 (1:1000 Abcam, UK), Smoothened (1:1000 Abcam, UK) and Beta-actin (1:5000 Bioworld, USA). The next day, the membranes were incubated with the appropriate HRP-conjugated secondary antibodies for 2 h at room temperature. Immunoreactive bands were visualized with chemiluminescence reagents that were provided with an ECL kit (Bioworld, USA). Blot intensity was detected using Image J software.

The testicular tissue samples were homogenized, and protein quantifications were performed using a BCA protein assay kit (Beyotime, Shanghai, China). Equal amounts of protein per specimen were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (0.2 μl or 0.45 μm) (Millipore Corporation, Bedford, MA, USA). The membranes were blocked with 5% fat-free milk for 2 h at room temperature, followed by incubation with the following antibodies at 4°C overnight: nuclear factor like-2 factor (Nrf2; Abcam 1 : 1,000), HO-1 (Abcam 1 : 1,000), quinone oxidoreductase-1 (NQO-1; Abcam 1 : 1,000), NF-κB (CST 1 : 1,000), necrosis factor alpha (TNF-α) (CST 1 : 1,000), interleukin-1 beta (IL-1β) (CST 1 : 1,000), interleukin-6 (IL-6) (CST 1 : 1,000), and beta-actin (Bioworld 1 : 10,000). After washing with Tris-buffered saline and Tween 20 (TBS-T), the blots were incubated with the horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (BioSharp, Technology Inc., China) at room temperature for 2 h. Finally, protein bands were detected using Image Lab Software and analyzed using Image-Pro Plus software. All the experiments were repeated at least 3 times.