Levels of c-myc and max proteins in 32D cell lines. (A) c-myc levels. From the indicated cell lines, 50 μg of protein was resolved by SDS-PAGE, Western blotted, and probed with a polyclonal anti–c-myc antibody.40 Lane 1 shows the level of endogenous c-myc protein in logarithmically growing 32D-neo cells. Lane 2 shows the disappearance of c-myc in the same cells after a 6-hour deprivation of IL-3. In lanes 3 through 14, the indicated cultures were deprived of IL-3 for 6 hours to deplete endogenous c-myc stores. Note the complete absence of endogenous c-myc protein in IL-3–deprived 32D-neo and 32D-max cells. Lanes 4, 7, 10, and 13 show that c-myc arising from the transfected expression plasmid was expressed at levels 2 to 3 times that of endogenous c-myc and was not downregulated after IL-3 deprivation. (B) max levels. The indicated cell lines were labeled with 35S-Translabel as previously described.18 After lysis in triple detergent radioimmunoprecipitation buffer, max proteins were immunoprecipitated with a polyclonal rabbit anti-max antibody, resolved by SDS-PAGE, and subjected to autoradiography. The arrow indicates the position of endogenous and overexpressed max(L).
