Immunoblotting Analysis of Cell Cycle Regulators

Cells were rinsed in ice-cold phosphate-buffered saline (PBS) and extracts prepared in lysis buffer (50 mM Tris HCl pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 10 mM Na β-glycerophosphate, 50 mM NaF, 5 mM Na Pyrophosphate, 270 mM sucrose and 1% Triton X-100) supplemented with protease inhibitor (Roche) and 1 mM phenylmethylsulfonyl fluoride. Lysates were subjected to SDS-PAGE and transferred to a PVDF membrane (ImmunolonP, Thermo). The following antibodies were used for immunoblotting: anti-rabbit and anti-mouse horseradish peroxidase-conjugated secondary antibodies (1:5000; Pierce); anti-human Hsp90 (1:1000; 610418, BD Biosciences), anti-SAMHD1 (1:1000; ab67820, Abcam), anti-CDK1 (9116) anti-CDK2 (2546), anti-phosphoCDK2 (Thr160; 2561), anti-CDK4 (D9G3E), anti-CDK6 (3136), anti-cyclin A2 (BF683), anti-cyclin D2 (D52F9), anti-cyclin D3 (DCS22), anti-p21 (2947) and anti-p27 (2552) all 1:1000 from Cell Signaling. Anti-phospho-SAMHD1 Thr592 was obtained by immunization of rabbit using a phosphorylated peptide as described before [53 (link)].