Protein inhibitor cocktail set 3

Logarithmically growing cells were harvested and frozen at −80°C. Pellets were resuspended in the same volume of HB2 buffer (50 mM HEPES/KOH at pH 7.5, 140 mM NaCl, 15 mM EGTA, 15 mM MgCl₂, 0.1% NP-40, 0.5 mM Na₃VO₄) containing protease inhibitors (1 mM dithiothreitol, 1 mM PMSF, 0.1% Protein inhibitor cocktail set III (Sigma), 0.1 ng/ml MG132 (Sigma), 10 U/ml TURBO DNase (Ambion)). Cells were broken using a Fast Prep machine (Thermo), briefly sonicated using the Biorupter and centrifuged to harvest the chromatin containing cell extract. Monoclonal anti-Flag M2 antibody (Sigma), anti-Myc 9E11 (Cell Signalling), anti-PK antibody (AbD Serotec/Bio-Rad) and anti-HA antibody (Covance) were used for pull down and detection of FLAG, Myc, PK and HA tagged proteins respectively. Anti-Cdc2 antibody (Santa Cruz) was used as a control. For protein complex immunoprecipitation, antibodies were conjugated with mouse IgG-coated Dynabeads (Life Technologies).

Logarithmically growing cells were collected and frozen at −80 °C. Pellets were resuspended in the same volume of HB buffer (50 mM HEPES/KOH at pH 7.5, 140 mM NaCl, 15 mM EGTA, 15 mM MgCl₂, 0.1% NP-40, 0.5 mM Na₃VO₄) containing protease inhibitors (1 mM dithiothreitol, 1 mM PMSF, 0.1% protein inhibitor cocktail set III (Sigma), 0.1 ng mL⁻¹ MG132 (Sigma), 10 U mL⁻¹ TURBO DNase (Ambion)). Cells were broken using a Fast Prep machine (Thermo), briefly sonicated using the Biorupter and centrifuged to collect the chromatin containing cell extract. Monoclonal anti-Myc 9E11 (2276, Cell Signalling) and anti-HA antibodies (901514, Covance/BioLegend) were used for pull down and detection of Myc and HA tagged proteins, respectively. Antibodies were conjugated with mouse IgG-coated Dynabeads (Life Technologies) overnight. Whole-cell extracts (WCEs) were incubated with the pre-coated beads for 1 h at 4 ˚C, washed, and then resuspended in SDS loading buffer and boiled. Samples were subjected to western blotting with anti-HA (1:1000), anti-Myc (1:5000), or anti-PK (1:4000, anti-V5 SV5-Pk2, MCA2892, Covance/Bio-Rad) antibodies. Anti-Cdc2 (sc-53, Santa Cruz) was used as a control (1:5000).