Cdk4 sc 260

Cells were treated as described in the corresponding section of Results and then lysed by boiling for 10 min in sample buffer (2% SDS, 10% glycerol, 10% β-mercaptoethanol, bromphenol blue and Tris-HCl, pH 6.8). Lysates were fractionated on polyacrylamide gels and transferred to nitrocellulose. The blots were probed with specific antibodies followed by secondary antibody then membranes were examined using the LI-COR Odyssey infrared imaging system (LI-COR Biotechnology, Lincoln NE). The AR (N20, sc-816 and H280, sc-13062; 1:1,000), HSP90 (sc-7947; 1:1,000), and Cdk4 (sc-260; 1:1,000) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The HSP70 (1776-1; 1:5,000), HSP40 (3532-1; 1:1,000) and ubiquitin (1646-1; 1:1,000) antibodies were purchased from Epitomics (Burlingame, CA). p23 (ab92503; 1:1,000) and Hop (ab126724; 1:1,000) antibodies were purchased from Abcam (Cambridge, MA). Akt (4691; 1:1,000) and HDAC6 (7558; 1:1,000) antibodies were purchased from Cell Signaling Technology (Danvers, MA). CDC37 (4222S; 1:1,000) antibody was purchased Biogot Biotechnology Co., Ltd (Shanghai, China). The β-actin antibody (1:10,000) was purchased from Sigma (St Louis, MO). The secondary antibody was conjugated with IRDye 680/800 (Millennium Science; 926–32221, 926–32210; 1:10,000). Uncropped western blots are shown in Supplementary Fig. 13.

Puromycin and G418 were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). NEM and doxorubicin was purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). CHX and Mg132 were obtained from MedChemExpress (Shanghai, China). The following antibodies were used for either western blotting or immunohistochemical analysis: FBXO22 (13606–1-AP, Protein-tech, Wuhan, China), CDK2 (SC-748, Santa Cruz Biotechnology, CA, USA), CDK4 (SC-260, Santa Cruz Biotechnology, CA, USA), cyclinE1 (EP435E, Abcam, Cambridge, MA, USA), and GAPDH (KC-5G4; Kang Chen Bio-tech, Inc., Shanghai, China). The following antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA): Akt (C67E7), phospho-Akt (Ser473) (D9E), Erk (137F5), p38 (D13E1), phospho-p38 (3D7), p70s6k (49D7), phospho-p70s6k (1A5), cyclin B1 (V152), cyclin D1 (92G2) and p21(12D1).

Protein expression levels were analyzed by standard Western blotting protocols. The following antibodies were used in this study: hBD3 antibody (500-P241) from PeproTech (Rocky Hill, NJ, USA); AKT (pan) (#4691), ERK 1/2 (#9102), phospho-AKT (#2965), phospho-ERK1/2 (#4376), p65 (#8242) and phospho-p65 (#3033) antibodies from Cell Signaling Technology (Danvers, MA, USA). Cyclin A (sc-596), cyclin E (sc-247), Cdk2 (sc-163) and Cdk4 (sc-260) from Santa Cruz Biotechnology (Dallas, TX, USA); β-actin (SP124) antibody from Sigma-Aldrich (USA). Cyclin D1/D2 was from R&D Systems (Minneapolis, MN, USA).

Roswell Park Memorial Institute-1640 (RPMI-1640) medium, penicillin-streptomycin solution, and trypsin-EDTA (0.05%) were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Tannic acid (TA; Cas. No: 1401-55-4), fetal bovine serum (FBS; 12003C), Zb (Z3902), and primary antibodies specific for SOX2 (MAB4423), OCT4 (MABD76), and NANOG (MABD24) were purchased from Sigma-Aldrich (Merck KGaA, St. Louis, MO, USA). Antibodies specific for β-actin (sc-47778), TRAIL (sc-8440), TRADD (sc-46653), Wnt5A (sc-365370), Casp8 (sc-81656), Bcl-2 (sc-7382), p21 (sc-756), cyclin E (sc-481), and CDK4 (sc-260) and secondary antibodies (anti-mouse (sc-516102) and anti-rabbit (sc-2357)) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The Wnt8A (H00007478-B01P) antibody was obtained from Abnova (Taipei City, Taiwan); pEGFR (#2234), β-Catenin (#9582), GSK-3β (#9315), Bax (#2772), Casp9 (#9502), C-Casp9 (#9505), Casp3 (#9662), C-Casp3 (#9661), cytochrome C (#11940), and p27 Kip1 (#3686) antibodies and a TCF/LEF Family Antibody Sampler Kit (#9383) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Finally, DR4 (ab8415), DR5 (ab8416), and cyclin D1 (ab6152) antibodies were purchased from Abcam (Cambridge, MA, USA).