TPA + IFN-γ costimulation induces modification of Myc. (A) Aliquots of untreated or stimulated U-937-myc-6 and U-937-GTB cells were in vivo labeled with ³²P-orthophosphate (upper panel) or ³⁵S-methionine (lower panel) as indicated whereafter cell lysates were immunoprecipitated with IG-13 pan-Myc antiserum. (B through G) ³⁵S-labeled lysates of untreated (B and F), TPA + IFN-γ- (C, D, and G) or TPA stimulated (E) U-937-myc-6 cells were immunoprecipitated with IG-13 pan-Myc antibodies and analyzed by 2D-gel electrophoresis as described in Materials and Methods. In (D) the antibody was blocked by incubation with recombinant c-Myc protein. In (F) and (G) the immunoprecipitates were phosphatase treated. The positions of v-Myc and c-Myc and of two unspecific spots are indicated by long and short arrows, respectively. (H) BK3A cells were labeled with ³⁵S-methionine, lysed in L-buffer and Myc:Max complexes immunoprecipitated using Myc-specific antibodies (5042). Treatment with alkaline phosphatase (PPase) in the presence or absence of β-glycerophosphate (β-GP) was performed as indicated. Max extracted in L-buffer (S) or bound to Myc (P) was analyzed by SDS-PAGE and fluorography.