A5547

Whole cell lysates were extracted using the RIPA lysate (P0013B, Beyotime) and quantified with a BCA protein assay kit. Equal amounts of protein samples were separated by SDS-PAGE and transferred onto the PVDF membrane. After blocking in 5% BSA, membranes were incubated with primary antibodies overnight at 4°C. Subsequently, immunoblots were incubated with secondary antibodies for 40 min at 37°C and visualized using Western ECL Substrate (E003, 7 Sea Biotech, Shanghai, China).
All antibodies were as follows: Occludin (1 : 1000; A2601, ABclonal), ZO-1 (1 : 1000; A0659, ABclonal), PGC-1α (1 : 1000; A17089, ABclonal), NRF-1 (1 : 1000; A5547, ABclonal), NRF-2 (1 : 1000; A0674, ABclonal), Tfam (1 : 1000; A13552, ABclonal), AMPK (1 : 1000; AF6423, Affinity), p-AMPK (1 : 1000; AF3423, Affinity), SIRT1 (1 : 1000; A11267, ABclonal), β-actin (1 : 2000; 60008-1-Ig, Proteintech, Wuhan, China), goat anti-mouse IgG antibody (1 : 10000; SA00001-1, Proteintech), and goat anti-rabbit IgG antibody (1 : 10000; SA00001-2, Proteintech).

Mouse testis tissues were collected at 24 h after radiation and lysed with RIPA lysis buffer. The protein concentrations were measured by the BCA protein detection kit. Western blotting was performed according to standard procedures. Briefly, equal amounts of the protein were separated by SDS‐PAGE, followed by transfer to polyvinylidene fluoride membranes (Bio‐Rad, USA). The membranes were incubated with antibodies against the proteins Bax (1:800, WL01637; Wanleibio, CHN), Bcl‐2 (1:500, WL01556; Wanleibio, CHN), β‐tublin (1:1000, 10094‐1‐AP; Proteintech), Tfam (1:2000, ab131607; Abcam, UK), PGC‐1α (1:1000, ab54481; Abcam, UK), and Nrf1(A5547; Abclonal, CHN) overnight at 4°C (all from Proteintech, USA). After being washed three times with TBST, the membranes were incubated with secondary antibody (HRP‐conjugated goat anti‐rabbit IgG, SA00001‐2, 1:5000; Proteintech, USA) for 1 h at room temperature. After final washes, the gray values of target bands were analyzed using Image J (version1.51; National Institutes of Health, USA) with β‐tublin as an internal control.