Klf12

Ovarian tissues were serially sectioned using formalin fixation and paraffin embedding. The sections were dewaxed in xylene, treated with gradient alcohol, and subsequently treated with 3% H₂O₂ for 20 min. The antigen was repaired at high temperature with sodium citrate buffer (pH 6.0) and subsequently incubated in the blocking solution for 30 min. Primary antibodies were incubated overnight at 4 °C using primary antibodies as follows: cleaved caspase-3 (1:200 dilution; Cell Signaling Technology) or KLF12 (1:500 dilution; Santa Cruz Biotechnology). The sections were then washed three times with Tris-buffered saline, and secondary antibody was added. The sections were stained with 3,3′-diaminobenzidine and counterstained with hematoxylin. Control sections were incubated with a nonspecific rabbit immunoglobulin G antibody (53 (link)).

Proteins were prepared as previously described [19 (link)], and the protein content was measured using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts (20 μg) of protein were separated by SDS-PAGE, and immunoblotting was performed with primary antibodies against FOXO1 (Cell Signaling Technology, Danvers, MA, USA, 1:1000), KLF12 (Santa Cruz Biotechnology, CA, USA, 1:2000) or GAPDH (Bioworld Technology, MN, USA, 1:10000), followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody and Flag-HRP (Sigma, 1:5000). The bands were detected using an enhanced chemiluminescence kit (Amersham Biosciences Corp., Piscataway, NJ, USA), and densitometric analysis of each band was performed with Quantity-one (Bio-Rad) software.

Proteins were extracted from cultured GCs or ovarian tissues as described previously (52 (link)). Briefly, protein concentrations were measured using BCA Protein Assay Reagent (Thermo Fisher Scientific). Proteins were separated using SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore). After incubation with primary antibodies and secondary antibodies of the corresponding species, exposures were recorded using a chemiluminescent horseradish peroxidase substrate kit (Millipore). The following antibodies were used: β-actin (1:10,000 dilution, catalog no.: AP0060; Bioworld Technology), KLF12 (1:500 dilution, catalog no.: sc-134373; Santa Cruz Biotechnology), SPHK1 (1:500 dilution, catalog no.: CY6962; Abways), cleaved caspase-3 (1:500 dilution, catalog no.: 9661S; Cell Signaling Technology), caspase-3 (1:1000 dilution, catalog no.: 9662S; Cell Signaling Technology), p-AKT (1:500 dilution, catalog no.: sc135650; Santa Cruz Biotechnology), and AKT (1:500 dilution, catalog no.: BS2987; Bioworld Technology), p-FOXO1(1:500 dilution, catalog no.: ab47326; abcam), and FOXO1 (1:2000 dilution, catalog no.: 2880S; Cell Signaling Technology).