Anti phospho cdc2 tyr15 antibody

To determine the phosphorylation level of Cdc28, total protein of the tested strains was separated by 12% SDS-PAGE and immunoblotted with an anti-phospho-Cdc2 (Tyr¹⁵) antibody (Cell Signaling Technology). The anti-Cdc2 antibody (Santa Cruz Biotechnology) was used as a negative control.

Protein preparation, immunoprecipitation and immunoblotting were performed as previously described [18 (link)]. Cell cultures were mixed with 7% trichloroacetic acid and put on ice for a minimum of 10 min. After centrifugation, the cell pellets were washed twice with cold acetone and dried. The cells were lysed in buffer A [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), 10% glycerol, 20 mM β-glycerophosphate, 0.1 mM Na₃VO₄, 10 mM p-nitrophenyl phosphate (p-NPP) and 10 mM NaF] containing 0.2% NP-40 and glass beads and then vortexed. Whole-cell extracts were mixed with 3 × SDS sample buffer and boiled for 5 min. The supernatants of these boiled lysates were separated by SDS-PAGE and then subjected to Western blotting. Anti-α-tubulin (B5-1-2) antibody was obtained from Sigma. Mouse anti-HA (12CA5) antibodies were acquired from Roche. Anti-PSTAIRE antibody was purchased from Santa Cruz. Anti-phospho-Cdc2 Tyr-15 antibody was purchased from Cell Signaling Technology. After incubation with HRP-conjugated secondary antibodies, proteins were detected using the ECL Plus Western Blotting Substrate (Thermo Scientific). The immunoblot band intensities were measured by Image J software (NIH).