Fig. 2

IκB kinases phosphorylate c-Myc at the N-terminal end. a Phosphorylation of full-length c-Myc by IKKα: HEK-293 cells were transfected with a flag-tagged constitutive active IKKα expression construct, followed by purification of the kinase with anti-flag beads and an in vitro kinase reaction with recombinant c-Myc. After the kinase reaction, samples were applied to SDS-PAGE and the dried gel was exposed to an x-ray film. Three independent samples with the substrate (+) are shown in comparison to a negative control (−). Unconjugated, free ³²P-ATP is seen at the front of the gel. b Protein alignment showing conservation of a putative IKK-target site (SGLCS motif) next to the known GSK3β phosphorylation site in diverse species. c Kinase assay showing IKKα and IKKβ-mediated phosphorylation of a c-Myc peptide (as specified below the gel) containing the putative IKK target site. d Peptide kinase assay with detection of the incorporated ³²P using a liquid scintillation counter. Flag-tagged IKKα or IKKβ was transfected into HEK-293 cells, followed by purification with flag-affinity matrix. Biotinylated peptides of IκB containing a known IKK phosphorylation site or a mutated, negative control (IκB-mut) and peptides containing the putative phosphorylation site of c-Myc or a mutated variant thereof (c-Myc-mut, sequences as indicated) were incubated with the kinases, followed by purification of biotinylated peptides on neutravidin-coated plates and measurement by scintillation counting