Figure 1
From: Phosphorylation of Puma modulates its apoptotic function by regulating protein stability
Exogenous Puma is phosphorylated on serine residues in HeLa cells in a BH3 domain-independent manner (a and c) HeLa cells were transfected with a plasmid encoding HA-Puma (a) or Flag-Puma (c) and incubated in the presence of caspase inhibitor BAF for 16–20 h before lysis. Overexpressed Puma was immunoprecipitated using anti-HA antibody and protein A beads or anti-Flag beads. Immunoprecipitated Puma was detected using an anti-Puma N-terminus antibody. For analysis of Puma phosphorylation, cells were incubated for 4 h with 32P-orthophosphate before lysis. After SDS-PAGE, gels were stained with Coomassie to visualise protein and dried. 32P-labelled HA-Puma (b, upper panel) and Flag-Puma (c, upper panel) were detected using a phosphorimager system. HA-Puma-WT was run in duplicate in panel b. (d) 32P-labelled HA-Puma was transferred to PVDF membrane. The radioactive Puma band was excised from the membrane and amino acids were obtained by acid hydrolysis. 32P-labelled amino acids were resuspended in 10 μl milliQ water including phosphoamino acid standards and this sample was run on a 1D-TLC plate. Phosphoamino acid standards were visualised by ninhydrin staining, and 32P-labelled amino acids were detected using a phoshorimager system
