Figure 5

The non-phosphorylatable Puma serine 10 mutant accumulates in a manner independent of caspases, apoptosis and autophagy. (a) DKO MEFs were lysed 24 h after transfection with the indicated constructs. Proteins were resolved by SDS-PAGE and Puma protein was detected using an antibody directed against human Puma. (b) qRT-PCR analysis of DKO MEFs transfected for 20 h with the indicated constructs. The amount of Puma mRNA was normalised in each sample against 18S RNA. (c) WT or ATG-5 KO MEFs were transfected with either WT or S10A Puma constructs in the presence or absence of pan-caspase inhibitor zVAD-fmk. At 24 h after transfection, cells were lysed and Puma expression analysed by western blot using a Puma N-terminal antibody. (d) WT and DKO MEFs were transfected with non-apoptotic Puma lacking a functional BH3 motif (ΔLRR) containing either the wild-type serine at position 10 or an alanine substitution. At 24 h after transfection, cells were lysed and Puma expression was analysed by western blot analysis using anti-Puma antibodies