Ab33889

The collected T24 cells were lysed using radio-immunoprecipitation assay solution (SY4680, YITA Biotechnology, Beijing, China), and protein concentration was examined by bicinchoninic acid kits (P0012, Beyotime). Following separating with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, proteins were electrotransferred to polyvinylidene fluoride membranes. Following blockade with 5% skim milk for 2 h, membranes were probed overnight at 4 °C with following rabbit anti-human primary antibodies: matrix metalloproteinase MMP-2 (1/10,000, ab92536, Abcam, Cambridge, UK), light chain 3B (LC3B; 1/50, ab192890), p62 (1/1000, ab207305), phosphorylated p53 (p-p53; 1/1000. ab33889), p53 (1/2000, ab179477), p-mTOR (1/2000, ab109268), mTOR (1/10,000, ab134903), and β-actin (1/1000, ab8227), followed by 1-h incubation with the horseradish peroxidase-labeled goat anti-rabbit secondary antibody IgG (1/50,000, ab205718) at room temperature. Afterward, the images were developed with enhanced chemiluminescence working solution (E412-01, Vazyme). The bands were quantified utilizing Image-Pro Plus 6.0 (Media Cybernetics), with β-actin as an internal reference.

To evaluate protein localization and intensity, immunohistochemistry and immunofluorescence were performed according to previously described methods [26 (link), 27 (link)]. Ovarian tissue slides were dewaxed in xylene and graded series of ethanol solutions. Slides were then fixed in 4% paraformaldehyde and blocked with 3% bovine serum albumin (BSA) at 37°C. The sections were incubated overnight at 4°C with antibodies against p-IRE1α (1: 200, ab124945, Abcam, Cambridge, UK), XBP1 (1: 100, WL00708, Wanleibio, Shenyang, China), p-PI3K (1 : 200, AF3242, Affinity, USA), p-AKT (1: 200, 4060T, CST, USA), p-p53 (1: 200, ab33889, Abcam, UK), p-NF-κB (1: 200, 3033S, CST, USA), and cleaved-caspase-3 (1: 200, 66470-2-Ig, Proteintech, USA). All secondary antibodies were diluted (1 : 2000) and incubated at 25°C for 2 h. The sections were processed according to the avidin-biotinylated-peroxidase complex and DAB staining techniques, followed by observation under an optical microscope. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (1 : 2000, C1002, Beyotime, China) for 30 min and photographed using an Olympus laser scanning confocal microscope (FV3000).

Cells were first lysed (Solarbio, Beijing, China) and then denatured in SDS buffer to obtain total protein. The protein was separated on a 10% SDS-polyacrylamide gel (30 mg per lane) and transferred to a PVDF membrane (Merck Millipore, MA, USA). After blocking in 5% skim milk for 1 h, the membrane was incubated with primary antibody overnight at 4 °C and then incubated with the secondary antibody (1:15,000, GE Healthcare, Cambridge, UK) for 1 h at room temperature. Then, visualization was performed with ECL chemiluminescence reagent (Merck Millipore, MA, USA).
The antibodies used in this study were as follows: CRABP2 (1:1,000, ProteinTech, Cat. # 10225-1-AP), BAX (1:1,000, Abcam, ab32503), BCL-2 (1:1,000, Abcam, ab32124), cleaved caspase-3 (1:1,000, Abcam, ab32042), PARKIN (1:1,000, ProteinTech, Cat. # 14060-1-AP), ATR (1:1,000, Abcam, ab2905), p-ATR (1:1,000, Abcam, ab178407), ATM (1:1,000, Abcam, ab32420), p-ATM (1:1,000, Abcam, ab81292), P53 (1:1,000, Abcam, ab32389), p-P53 (1:1,000, Abcam, ab33889), TET1 (1:1,000, Abcam, ab272900), DNMT3A (1:1,000, Abcam, ab188470), DNMT3B (1:1,000, Abcam, ab2851), P62 (1:1,000, ProteinTech, Cat. # 18420-1-AP), TOMM40 (1:1,000, Abcam, ab185543), α-tubulin (1:5,000, Abcam, ab7291), β-actin (1:5,000, ProteinTech, Cat. # 66009-1-Ig), GAPDH (1:20,000, ProteinTech, Cat. # 60004-1-Ig).