FIG. 4.

(A) Mutated c-Myc (Thr-58Ala) is constitutively hyperphosphorylated in PA682 BL cells. c-Myc was precipitated from PA682 protein extracts (500 μg) with anti-c-Myc (Ab-3) (lane 3). Parallel samples (250 μg of immunoprecipitated protein) were treated with increasing concentrations of PAP (lanes 4 and 5). Protein extracts from 293 cells transfected with p64 or p67 c-Myc were directly loaded for SDS-PAGE and used as size marker controls in the immunoblot analysis (lanes 1 and 2). IgH, heavy-chain immunoglobulin. Numbers on the right are molecular masses in kilodaltons. (B) In vitro phosphorylation of c-Myc reduces binding to α-tubulin. The N-terminal portion of c-Myc expressed as a GST fusion protein substrate (GST-Myc II) was treated with CKII and MAP kinase (MAP) and incubated with HL60 cell lysate. Precipitated proteins were resolved by SDS-PAGE and immunoblotted with α-tubulin and c-Myc antibodies.