Pten sc 7974

C3H10T1/2 cells were bought from ATCC (Manassas, VA, USA) and cultured with complete Dulbecco's modified Eagle's medium, which containing 10% fetal bovine serum (FBS), 100 μg/ml streptomycin, and 100 U/ml penicillin. Besides this, cells were saturated at 37°C and 5% CO₂. Primary antibodies against PTEN (sc-7974), Runx2 (sc-390715), OPN (sc-21742), and GAPDH (sc-47724) were ordered from Santa Cruz Biotechnology; Wnt10b (ab70816), CREB (ab32515), and p-CREB (ab32096) were ordered from Abcam; Smad1/5/9 (13820S) was ordered from CST; and p-Smad1/5/9 (AF8313) was bought from Affinity Biotech.

For the in vitro study, LNCaP cells were treated with EGCG (20 µM) + Q (2.5 µM), Arc (1 µM), EGCG+Q+Arc, or the control; WPE1-NA22 cells were treated with EGCG (20 µM) + Q (2.5 µM), Arc (0.5 µM), EGCG+Q+Arc, or the control, for 24 h. The cells were harvested and total protein was extracted using an RIPA lysis buffer (Santa Cruz, CA, USA). For the animal study, total protein was extracted from the prostate/tumor tissues using the RIPA lysis buffer (Santa Cruz). The procedure for the Western blot analysis was described previously [38 (link)]. Briefly, 50 μg of protein was separated on a 4–12% Bis-Tris gel (Invitrogen), electrotransferred to nitrocellulose membranes, and blocked in 5% nonfat milk. The membranes were incubated with primary antibodies for the detection of AR (sc-7305, 1:500 dilution), PTEN (sc-7974, 1:500 dilution) (Santa Cruz Technology, Santa Cruz, CA, USA), Akt (4685, 1:1000 dilution), and p-Akt (4058, 1:1000 dilution) (Cell Signaling Technology, Danvers, MA, USA), respectively. GAPDH was used as a loading control.

Following PGRMC1 siRNA transfection and AG-205 treatment, cells were harvested, and protein was extracted using the mammalian Protein Extraction Reagent (mPER) according to the manufacturer’s protocol (Thermo Scientific, US). Protein concentration was quantitated and determined using the Pierce^TM BCA Protein Assay Kit (Thermo Scientific, US). Equal amounts of protein were loaded and separated by SDS-PAGE using Mini-Protean TGX polyacrylamide gels (Bio-Rad). Proteins were then transferred onto PVDF membranes, blocked with 5% BSA in 1 × TBST and probed with the respective primary antibodies followed by the appropriate secondary antibody, and were subsequently visualised using an enhanced chemiluminescence kit (Thermo Scientific, US). Primary antibodies used for the experiments were as follows: AKT (14702S), p-AKT (4060S), p-EGFR (3777S), mTOR (2972S), p-mTOR (2971S), PGRMC1 (13856S), Cl. Caspase 3 (9661S), Cl. PARP (5625S), PARP (9532S), BAX (2772S), CDK4 (12790P) and Cyclin D1 (2978P), all of which were obtained from Cell Signaling Technology; PTEN (sc-7974) and p-EGFR (sc-101665), purchased from Santa Cruz Biotechnology; EGFR (ab-2430-1) from Abcam; β-actin (A1978) from Sigma-Aldrich. ImageJ software was used to quantify protein levels detected.