Rabbit anti pk2 antibody

The testicular tissue was lysed on ice with radioimmunoprecipitation assay lysis buffer (Cwbio, Taizhou, China). Subsequently, 20–40 μg of protein was electrophoresed on an 8%–12% (w/v) sodium dodecyl sulfate-polyacrylamide gel gradient and transferred to a nitrocellulose membrane. The membranes were blocked with 5% (w/v) nonfat milk for 1 h in Tris-buffered saline (TBS) and subsequently incubated with the following primary antibodies: rabbit anti-PK2 antibody (1:200, Abcam, Cambridge, MA, USA), rabbit anti-cleaved-caspase-3 antibody (1:1000, Cell Signaling Technology [CST], Boston, MA, USA), rabbit anti-caspase-8 antibody (1:1000, CST), rabbit anti-Bax antibody (1:1000, CST), rabbit anti-Bcl-2 antibody (1:1000, CST), and mouse anti-β-actin antibody (1:500, Boster, Wuhan, China). Immunoreactive bands were detected using electrochemiluminescence (ECL; Pierce, Waltham, MA, USA).

The rat was perfuse-fixed with 300–350 ml 4% formaldehyde solution (Servicebio, Wuhan, China) after 200 ml normal saline via a perfusion needle in the left ventricle of the heart for 45 min. Then, the testis was removed and immersion fixed in the same fixative for 4–6 h. Testicular sections were stained with hematoxylin–eosin (H and E; Solarbio, Beijing, China) to determine the histological changes. To assess the location of PK2 and PKR1 in the rat testes, the sections were subjected to antigen retrieval by heating in citric acid buffer (0.018 mol l⁻¹ citric acid monohydrate and 0.082 mol l⁻¹ trisodium citrate dihydrate [Sinopharm Chemical Reagent Co.], pH 6.0) at 95°C for 15 min. After natural cooling, the sections were incubated in PBS containing 3% (v/v) H₂O₂ for 20 min to inhibit endogenous peroxidase activity. Then, the testicular sections were blocked with 2.5% (w/v) nonfat milk for 10 min and incubated with the primary antibodies: rabbit anti-PK2 antibody (1:200, Abcam) and rabbit anti-PKR1 antibody (1:500, Alomone, Jerusalem, Israel) overnight at 4°C. The EnVision™ Detection Kit (Dako, Copenhagen, Denmark), based on the diaminobenzidine (DAB) working principle, was used to detect the expression of PK2 and PKR1. The signal was detected in an optical microscope (Olympus).

The GC-2 cells were seeded on a cover slip and fixed with cooled 4% (w/v) formaldehyde at 4°C for 20 min. After blocking with 5% (v/v) normal goat serum (Beyotime) at room temperature for 1 h, the cells were incubated with the rabbit anti-PK2 antibody (1:200, Abcam) or rabbit anti-PKR1 antibody (1:500, Alomone) at 4°C overnight. After the cells were washed twice with PBS, the secondary antibody, goat anti-rabbit IgG H&L (FITC; 1:500, Abcam), was incubated with the cells at room temperature for 1 h. The cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; biofroxx, Einhausen, Germany). The fluorescence intensity was detected with a fluorescence microscope (Olympus).