C myc

We made the bacterial expression plasmids pET-28B-huOct4, pET-28B-huSox2, pET-28B-huKlf4, and pET-28B-huMyc encoding the full length human O, S, K, M, respectively, fused to an N-terminal 6X histidine tag. The recombinant proteins were expressed in E. Coli Rosetta (DE3) pLysS (Novagen # 70956-3) and purified using a nickel charged column under denaturing conditions The mammalian expressed human OSKM recombinant proteins were obtained from OriGene (Oct4 #TP311998, Sox2 #TP300757, Klf4 #TP306691, c-Myc #TP301611). See Extended Experimental Procedures for more details of this and following sections.

Purified c-MYC (5 μg, Origene, 301611) in 50 μL PBS was incubated 30 min at room temperature either with DMSO vehicle or EN4 (50 μM). The DMSO control was then treated with light iodoacetamide while the EN4 treated sample was incubated with heavy iodoacetamide for 1 h each at room temperature (200 μM final concentration, Sigma-Aldrich, 721328). The samples were precipitated by additional of 12.5 μl of 100% (w/v) trichloroacetic acid and the treated and control groups were combined pairwise (10 μg total) before cooling to −80 °C for 2 h. The combined sample was then spun for at max speed for 10 min at 4 °C, supernatant was carefully removed and the sample was washed with ice-cold 0.01 M HCl/90% acetone solution. The pellet was resuspended in 4 M urea containing 0.1% Protease Max (Promega, V2071) and diluted in 40 mM ammonium bicarbonate buffer. The samples were reduced with 10 mM TCEP at 60 °C for 30 min. The sample was then diluted 50% with PBS before sequencing grade trypsin (1 μg per sample, Promega, V5111) was added for an overnight incubation at 37 °C. The next day, the sample was centrifuged at 13,200 r.p.m. for 30 min. The supernatant was transferred to a new tube and acidified to a final concentration of 5% formic acid and stored at −80 °C until mass spectrometry analysis.