Phospho prb

Western blot (WB) and immunoprecipitation (IP) analyses were performed as described previously [22 (link)]. The following antibodies were used in this study: Primary antibodies included phospho-AKT at S473 (#4060), phospho-AKT at T308 (#13038), phospho-Foxo3α at Thr32 9 (#9464), phospho-AMPKα1 at Thr172 (#2535), phospho-p70S6K at Thr389 (#9206), phospho-SIN1 at Thr86 (#14716S), phospho-NDRG1 at Thr346 (#5482), phospho-p53 at Ser6 (#9285), Ser9 (#9288), Ser15 (#9284), Ser20 (#9287), and Ser37 (#9289), phospho-pRb at Ser807/811 (#9308), mTOR (#2972), Raptor (#2280), Rictor (#2140), TSC2 (#6935), 4EBP1 (#9452), and phospho-4EBP1 at Thr37/46 (#2855) (Cell Signaling Technology, Danvers, MA, USA); PTEN (#SC-7974), MDM2 (#SC-965), phospho-PKCα at Ser657 (#SC-12356), and p21 (#SC-397) (SantaCruz, Dallas, TX, USA); p53 (#NCL-L-p53-DO7) (DO7, Leica, Milton Keynes, UK); Actin (#G043) (Applied Biological Materials Inc.); Flag (#A8592), and Catalase (#C-0979) (Sigma); Myc (#OP10) (Calbiochem, CA, USA).

For protein extraction, the assays were performed with 10-cm cell culture plates, and the cells were seeded at 500,000 cells per dish. Briefly, the cells were lysed using NETN buffer [8 (link)] supplemented with phosphatase inhibitors [8 (link)] and followed by three cycles of freezing (in liquid nitrogen) and thawing (in a 37 °C water bath). The protein extracts were separated by SDS-PAGE. The primary antibodies used for immunodetection were for MIG6 (Proteintech, 11630-1-AP), panAKT (Cell Signaling, 4685S, lot 6), AR (Biogenex, 256M), β-Actin (Abcam, ab6276, GR3324554-1), p-AKT (S473) (Cell Signaling, 4058S, lot 14), pRb (Abcam, ab6075, lot 821737), phospho-pRb (Cell Signaling, 9308, lot 13), panS6 (Cell Signaling, 2217S, lot 10), and p-S6 (S235/236) (Cell Signaling, 2211S, lot 23). Horseradish peroxidase-conjugated anti-mouse IgG (Cell Signaling, 7076S, lot 32) or anti-rabbit IgG (Cell Signaling, 7074S, lot 28) were used as secondary antibodies. The detection was performed by ImageQuant^TM LAS 4000 (GE Healthcare Bio-Sciences AB, Chicago, Illinois, U.S.). Quantification of bands was performed via the LabImage D1 program.

Western blotting was performed as previously described (28 (link)). Each membrane was blocked in 0.1% Tris buffered saline with Tween 20 (TBST) containing 5% bovine serum albumin and incubated with the indicated primary antibodies against phospho-p53, p21, phospho-pRb, TLR3, JAK1, phospho-STAT1 (Cell Signaling Technology, Danvers, MA), IL-6, p16, EZH2, CyclinD1 (Abcam, Cambridge, UK), PCNA (Biosciences, San Jose, CA), BMI1 (Active Motif, Carlsbad, CA), survivin (Genetex, Irvine, CA), and β-actin (Sigma-Aldrich). The membranes were then incubated with the corresponding peroxidase-conjugated secondary antibodies for 1 h at room temperature. The blots were developed using the Advansta Western Bright ECL HRP Substrate Kits (Advansta, San Jose, CA) and detected using a C-DiGit Blot Scanner (LI-COR, Lincoln, NE). The signal of each band was quantified using Image J densitometry software (Version 1.6, National Institutes of Health, Bethesda, MD) for densitometric evaluation. The targeted protein levels were normalized to the level of β-actin.