Flag m2 and β actin

To obtain total lysates, cells were lysed in RIPA buffer (50 mM Tris pH8.0, 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.5% SDS, protease inhibitors) on ice, followed by 10 min of sonication by Bioruptor. For IP, cells were lysed with buffer containing Tris-HCl (50 mM, pH8.0), NaCl (150 mM), Triton X-100 (0.5%), glycerol (10%), EDTA (1 mM) and protease inhibitors for 15 min on ice. The lysates were then incubated with Flag-M2 agarose (Sigma-Aldrich) or anti-JMJD2A (NeuroMab) at 4°C overnight. The precipitated protein complexes were washed four times next day. Proteins were resolved in SDS-PAGE for western blotting. Western blot images were taken by FluorChem E system and analyzed by AlphaView SA software. Antibodies used include: KDM4A (NeuroMab), Flag-M2 and β-actin (Sigma-Aldrich), HA (Covance), cyclin B1 and E2F1 (Santa Cruz), p-H3S10 (Millipore), PDK1 and PDK3 (Abcam). GST antibody was purified from rabbit antiserum generated against a GST-tagged antigen.

For immunoprecipitation, cells were gently detached by incubation in PBS, pelleted and lysed with FLAG IP buffer (50 mM Tris HCI, pH 7.4, 15 0mM NaCl, 1 mM EDTA and 1% TRITON X-100) in the presence of protease inhibitors (Complete EDTA free tablets; Roche, Indianapolis, IN). Lysate protein concentrations were determined by Pierce™ BCA (Thermo Scientific, Rockford, IL) and brought to equal protein concentration. Lysates were subjected to immunoprecipitation using Anti-FLAG® M2 Magnetic Beads (SIGMA-ALDRICH, St. Louis, MO). Briefly, lysates were incubated with beads for 2 hr (at RT) to overnight (at 4°C). Beads were washed 5x with lysis buffer and proteins eluted with 3x-FLAG® Peptide, 100 μg/ml, for 1 hr at RT. Cells used in degradation assays were lysed in SET Buffer (1% SDS, 50 mM Tris HCL, pH 7.4, 1 mM EDTA); lysates were thoroughly denatured by boiling for 5 min. Lysates and immunoprecipitation samples were resolved by SDS-PAGE on 4-10% Criteron™ TGX™ gels (Bio Rad, Hercules, CA) per manufacturer's recommendations and transferred to PVDF membrane (EMD Millipore, Billerica, MA). The following antibodies were used: FLAG® M2 and β-actin (Sigma Aldrich), HA.11 and 9E11(c-Myc)(Covance), and DDB1 and VprBP/DCAF1 (Abcam).