Figure 6

WT-Puma is degraded faster than non-phosphorylatable Puma S10A. (a) Puma is degraded by the proteasome. DKO MEFs infected with Myc-tagged wild-type Puma were treated with cycloheximide either in the absence or presence of the proteasome inhibitor MG132. At the indicated time periods, cell lysates were prepared and analysed by western blotting with antibodies against Puma and actin. (b) DKO MEFs were transfected with Myc-tagged Puma constructs as indicated. Cells were pulsed for 1 h with 50 μCi ³⁵S-labelled methionine/cysteine before a 4 h chase step in medium containing excess cold methionine/cysteine. Cells were lysed at the indicated time points after the pulse and Myc-tagged Puma immunoprecipitated. Puma expression was tested by western blot (b, lower panel) and the amount of ³⁵S label incorporated into Puma determined using a phosphorimager system. (c) Intensities of ³⁵S-labelled-Puma from four separate experiments were quantified by measuring band intensity using ImageJ (NIH, Bethesda, MD, USA) and are expressed as a percentage of the band intensity of WT or S10A at t=0. ^**P<0.01