Western blot

Ovarian tissues and cultured cells were homogenized in lysates [RIPA lysis buffer (CW2333S; CWBIO, China): PMSF (P0100; Solarbio, China) = 94:6] on ice for 30 min and subsequently centrifuged at 12,000 rpm for 20 min at 4°C. Supernatants were transferred to a clean 1.5 ml tube, and protein concentration was measured by BCA Protein Assay Kit (CW0014; CWBIO, China). Proteins were denatured by boiling at 100°C for 10 min and then separated by 10% SDS-PAGE, electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes, and blocked in PBST [phosphate-buffered saline (PBS) with 0.1% Tween-20] containing 5% skim milk powder for 2 h. After washing with PBST, the membranes were, respectively, incubated with antibodies against PKM2 (1:1,000, #4053; Cell Signaling Technology, USA), HK2 (1:1000, A0994; ABclonal, China), LDHA (1:1000, #3558; Cell Signaling Technology, USA), SIRT2 (1:1000, A12575; ABclonal, China), and GAPDH (1:1,000, #97166; Cell Signaling Technology, USA) overnight at 4°C. The membranes were further incubated with HRP-conjugated goat anti-mouse or goat anti-rabbit IgG (H+L) (1:5,000, SA00001-1/2; Protein Tech Group Inc., USA) for 2 h at room temperature. Finally, eECL (CW0049M, CWBIO, China) and the Tanon-5500 Chemiluminescence Imaging System were used to detect the chemiluminescence of protein bands.