Anti pten

Total cellular proteins were extracted using cell lysis buffer containing protease inhibitors (Boster) and phosphatase inhibitors (Biosharp) for western blotting and IP (Beyotime). The protein content of each sample group was determined using the BCA protein quantification kit (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, 20 μg of protein from each group of samples was separated on 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. Then, 5% skim milk powder was used to seal at room temperature for 1.5 h. After sealing, the PVDF membrane was treated with primary antibodies (Anti-phospho-PIK3CG (Bioss, Beijing, China, 1:2000), Anti-PTEN (Bioss, 1:2000), Anti-AKT1+2+3 (Bioss, 1:2000), Anti-phospho-AKT1+2+3 (Bioss, 1:2000), Anti-phospho-AKT3 (Bioss, 1:2000), GAPDH Antibody (Affinity, Liyang, China, 1:2000), and secondary antibodies (Goat Anti-Rabbit IgG H&L (HRP), Abcam, Cambridge, England, 1:5000). Photographic observations were made using an ultrasensitive ECL chemiluminescent substrate (Biosharp). Western blot results were analyzed using ImageJ v1.54d software (National Institute of Health, Bethesda, MD, USA).

CMT-U27 and CF41.Mg cells were cultured on gelatin-coated coverslips. Cells were fixed with 2% paraformaldehyde for 20 min at 4 °C and permeabilization using 0.5% Triton X-100 in PBS for 10 min. Blocking was performed using 5% animal serum (donkey or goat) in 2% bovine serum albumin in PBS for 1 h at room temperature. Subsequently, the cells were treated with primary antibodies against anti-ERβ (1:200; Abcam), anti-ERα (1:200; Invitrogen), anti-p-PI3K (1:200; Affinity Biosciences), and anti-PTEN (1:200; Bioss) in a blocking solution at 4 °C overnight. The cells were incubated with Alexa Fluor^TM 594 conjugated goat anti-mouse IgG (A-11005; 1:1000; Invitrogen) or Alexa Fluor^® 488 conjugated goat anti-rabbit IgG (ab150077; 1:1000; Abcam). The cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The cells were then mounted using mounting medium (Dako, Carpinteria, CA, USA) and images were captured using a THUNDER Imager 3D Live Cell & 3D Cell Culture System (Leika Microsystems, Wetzlar, Germany). The mean fluorescence intensity for each channel in the three distinct regions was quantified using ImageJ software (version 1.52a). The fluorescence intensity was expressed relative to that of the control.