Fbxo22 13606 1 ap

Cells were collected and lysed using Qproteome™ Mammalian Protein Prep Kit (Qiagen, Hilden, Germany). After centrifugation at 13,800×g for 10 min, the protein content in the supernatant was determined using the BCA protein assay kit (Bio-Rad, Shanghai, China). Equal amounts of protein were boiled by adding 4× sample loading buffers for 10 min at 100°C and resolved using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Antibodies against α-tubulin (11224-1-AP), CDK4 (11026-1-AP), and FBXO22 (13606-1-AP) were purchased from Proteintech (Rosemont, IL, USA), DNTM1 (ab188453) were purchased from Abcam (Cambridge, MA, USA), and PTEN (9188S) and ubiquitin (3936S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

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).

Cells were harvested and lysed with RIPA buffer (50 mM Tris–HCl, pH 7.6; 150 mM NaCl; 1 mM EDTA; 1% NP-40; 1% protease inhibitor cocktail; 1 mM PMSF), centrifuged at 17,000 g for 10 min at 4 °C. For immunoprecipitation of Flag-tagged proteins, supernatants were incubated with anti-Flag M2 Affinity Gel (SigmaAldrich) at 4 °C overnight. Otherwise, supernatants were incubated with indicated antibodies overnight and protein A/G-agarose beads (Santa Cruz, CA) for 4 h at 4 °C. The precipitates were washed three times with PBST buffer (PBS with 0.1% Tween-20), boiled in SDS-sample buffer, and subjected to immunoblotting analysis. For the protein expression analysis, standard western blotting was carried out with the following antibodies used: LKB1 (#3050), AMPKα (#2532), P-AMPKα (#2535), Raptor (#2280), P-Raptor (#2083), ACC (#3662), P-ACC (#11818), SKP1 (#12248), P-P70S6K (#9234), P70S6K (#2708), MO25α (#2716) were purchased form Cell Signaling Technology; FBXO22 (13606-1-AP) was purchased from Proteintech Group; Ubiquitin-K63 (EPR8590-448), NEDD8 (ab81264) were purchased from Abcam Technology; HA (H6908), Flag (A8592) and β-actin (A5316) were purchased from Sigma-Aldrich.