Monoclonal anti cdc2 p34 pstaire

Approximately 10⁸ cells were collected, resuspended in 100 μL water and, after adding 100 μL 0.2 M NaOH, they were incubated for 5 min at room temperature. Cells were collected by centrifugation, resuspended in 50 μL sample buffer, and incubated for 5 min at 95 °C. Extracts were clarified by centrifugation, and equivalent amounts of protein were resolved in an SDS-PAGE gel and transferred onto a nitrocellulose membrane. The primary antibodies used were monoclonal anti-HA peroxidase 3F10 antibody (Roche Diagnostics, Cat. No: 12013819001) diluted 1:5000, monoclonal anti-c-myc 9E10 antibody (Roche Diagnostics, Cat. No: 1667149) diluted 1:5000, monoclonal anti-GFP (Roche Diagnostics, Cat. No: 11814460001) diluted 1:5000, monoclonal anti-FLAG M2 (Sigma-Aldrich, Cat. No: F3165) diluted 1:10000 and monoclonal anti Cdc2 p34 (PSTAIRE) (Santa Cruz Biotechnology Inc., Cat. No: SC-53) diluted 1:2000. Blots were developed with anti-mouse IgG and anti-rabbit IgG Horseradish Peroxidase conjugate (Thermo Fisher Scientific, Cat. No: 170-6516 or 31460 respectively) diluted 1:20000 using the Supersignal West Femto Maximum Sensitivity Substrate (Thermo Scientific). Bands were quantified with a ImageQuant^TM LAS 4000mini biomolecular imager (GE Healthcare). Uncropped western blots are shown in Supplementary Figs. 9–15.

Chromatin immunoprecipitation analysis (ChIP), gene expression analysis by RT-PCR, protein co-immunoprecipitation assays and western blot analysis were performed as previously described (Gomar-Alba et al., 2017 (link)). In ChIP assays, Dynabeads Protein G magnetic beads were incubated with HA-probe (F-7) antibody (Santa Cruz Biotechnology; SC-7392) or monoclonal anti-GFP (Roche Diagnostics; 11814460001). The primary antibodies used in the western blot analysis were monoclonal anti-HA peroxidase 3F10 antibody (Roche Diagnostics; 12013819001) diluted 1:5000, monoclonal anti-GFP (Roche Diagnostics; 11814460001) diluted 1:5000 and monoclonal anti Cdc2 p34 (PSTAIRE; Santa Cruz Biotechnology; SC-53) diluted 1:2000. Blots were developed using anti-mouse IgG and anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies (1:20,000; Thermo Fisher Scientific; 170-6516 and 31460, respectively) and Supersignal West Femto Maximum Sensitivity substrate (Thermo Fisher Scientific). Bands were quantified using an ImageQuant LAS 4000mini Biomolecular Imager (GE Healthcare).