Anti flag m2 affinity agarose beads

Prior to immunoprecipitation, anti-Flag M2 affinity agarose beads (20 μL bead slurry, Sigma-Aldrich) were washed twice with cold TBS (Tris Buffered Saline, 500 μL; 20 mM Tris-Cl at pH 8.0, 150 mM NaCl). Lysates containing either wild type or mutant HDAC-Flag expressed proteins (1 mg total protein) were incubated with prewashed anti-Flag M2 agarose beads at 4°C overnight with rotation. After immunoprecipitation, beads were washed three times with lysis buffer (1 mL) containing high salt (500 mM NaCl). For the LSD1 binding assay, bound proteins were eluted with SDS buffer (20 μL; 100 mM Tris-Cl at pH 6.8, 4% SDS, 20% glycerol, 0.008% bromophenol blue) by boiling at 95°C for 5 min. For the p53 binding assay, bound proteins were eluted with TBS containing 3xFlag peptide (APEXBIO; 40 μL; 0.25 mg mL⁻¹ in TBS) for 30 min at 4°C. The eluted proteins were mixed with SDS loading dye (10 μL; SDS buffer containing 10% v/v β-mercaptoethanol) and boiled at 95°C for 2 min. As controls, lysates without immunoprecipitation (50 μg) were denatured with SDS loading dye (5 μL) by boiling at 95°C for 2 min. Proteins were separated by 10% SDS-PAGE, transferred to PVDF membrane (Immobilon P, Fischer Scientific), and immunoblotted with monoclonal FLAG^® M2 (F3165, Sigma), LSD1 (L4418, Sigma) or p53 (sc-126, SantaCruz Biotechnology) antibodies.

Cells were gently washed in PBS and then lysed using lysis buffer (1% NP-40, 50 mM Tris–HCl pH 7.5, 150 mM NaCl, complete protease inhibitor tablet, EDTA-free (Roche), 1 μM sodium orthovanadate, 1 mM sodium fluoride, and 0.1% β-mercaptoethanol). Lysed cells were scraped and transferred into a 1.5-ml microcentrifuge tube. Samples were incubated at 4°C for 20 min and then centrifuged for 15 min at 17,000x g at 4°C. Proteins (50 μg) were resolved on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad). Alternatively, the cleared cell lysates were incubated with anti-Flag M2 affinity agarose beads (Sigma-Aldrich), overnight at 4°C. Then the beads were washed three times with the lysis buffer then resuspended with sample buffer and then resolved on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad). Western blots were probed with the following antibodies: anti-Flag (M2) (Sigma-Aldrich), anti-c-NRAS (F155-277, Millipore), anti-NRAS (sc-519, Santa Cruz), anti-c-CBL (2747, Cell signaling), and anti-αtubulin (Millipore).

For in vitro kinase assays, N-terminally His6-tagged recombinant HDHB-EGFP (aa 994-1087 from HDHB) and RbC (aa 771-928 of Rb) (Adams et al., 1999 (link); Rubin et al., 2005 (link)) were purified by cobalt-sepharose affinity chromatography (GE Sepharose cat#17-0575-01) and eluted with 200 mM imidazole. Histone H1 protein (EMD Millipore) was used as a general substrate for Cdk. Human cyclin-Cdk fusion complexes were purified from yeast cells using a FLAG-tag method, modified from a previous protocol used for HA-tag purification (McCusker et al., 2007 (link)). Briefly, N-terminally tagged cyclin-Cdk fusions were cloned into 2-micron vectors using a glycine-serine linker (Rao et al., 1999 (link)) and overexpressed from the GAL1 promoter. The overexpressed 3xFLAG-tagged cyclin-Cdk complexes were then purified by immunoaffinity chromatography using anti-FLAG-M2 affinity agarose beads (Sigma-Aldrich A2220) and eluted with 0.2 mg/mL 3xFLAG peptide (Sigma-Aldrich F4799). We note that similar Cdk/cyclin fusions have been able to restore wildtype function in vivo (Chytil et al., 2004 (link)).