Ab179450

Ovarian tissues were washed thrice with pre-cooled PBS buffer and added with RIPA lysis buffer (Beyotime). A BCA protein assay kit (Beyotime) was used to evaluate protein concentration, after which the corresponding volume of protein was mixed with loading buffer (Beyotime) and heated in a boiling-water bath for 3 min. Electrophoresis was performed at 80 V for 30 min, and turned to 120 V for additional 1–2 h after bromophenol blue entered separation gels. The protein was blotted onto membranes in an ice-bath at a current of 300 mA for 60 min, after which the membranes were rinsed with washing buffer for 1–2 min and blocked in blocking buffer at room temperature for 60 min or at 4°C overnight. Incubation with anti-KLOTHO (PA5-99961, 1:1,000; Thermo Fisher Scientific), anti-FOXO1 (ab179450, 1:1,000; Abcam), anti-Bcl-2 (ab196495, 1:1,500; Abcam), or anti-Bax (ab32503, 1:1,000; Abcam) was performed on a shaker at room temperature for 1 h. The membrane was washed thrice with washing buffer for 10 min each, incubated with goat anti-rabbit immunoglobulin G (IgG) for 1 h at room temperature, and washed thrice for 10 min each. A chemiluminescence imaging system (Bio-Rad) was used to visualize the immunoblots after developer was added dropwise onto the membranes. All experiments were repeated in triplicate.

Mouse tumor tissue homogenate was lysed with the enhanced radio-immunoprecipitation assay (RIPA) lysate (Boster) containing a protease inhibitor for 20 min and centrifuged at 4°C at 3000 g for 20 min and isolation of the supernatant. The protein concentration in the supernatant was detected using the BCA protein quantitative kit. The protein content was isolated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The isolated protein content was transferred onto polyvinylidene fluoride (PVDF) membranes. A membrane blockade was induced using 5% bovine serum albumin (BSA) at room temperature for 2 h to block any nonspecific binding, prior to incubation with the primary antibody FOXO1 (ab179450, Abcam) at 4°C overnight. After a rinse with the tris buffered saline-Tween20 (TBST), the sample was supplemented with horseradish peroxidase (HRP) labeled secondary antibody and incubated for 1 h at room temperature. The enhanced chemiluminescence (ECL) reagent (EMD Millipore, Bedford, MA, USA) was added for development. Image Pro Plus 6.0 (Media Cybernetics Inc., Silver Springs, MD, USA) was used to quantify the gray scale of bands in each group in WB images, with β-actin serving as an internal parameter. Each experiment was conducted three times independently.

The cells or exosomes were lysed, and the isolated proteins were denatured in loading buffer after concentration detection. The separation of proteins was carried out by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis and then transferred to the polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were blocked and incubated with antibodies containing anti-CD63 (ab134045, 1:2000, Abcam, Cambridge, MA, USA), anti-CD81 (ab109201, 1:2000, Abcam), anti-TSG101 (ab125011, 1:3000, Abcam), anti-Calnexin (ab92573, 1:20000, Abcam), anti-FOXO1 (ab179450, 1:2000, Abcam) and anti-GAPDH (ab9485, 1:2500, Abcam) antibodies at 4°C overnight. After washing with TBST, the membranes were incubated with secondary antibody goat anti-rabbit IgG (ab6721, 1:5000, Abcam) for 2 h. Subsequently, the bands were visualized by the enhanced chemiluminescence (ECL) reagent (Millipore).