Fig. 3.

CaMKIV could phosphorylate PHB2 on consensus phosphorylation site. (A) Wild type or CaMKIV consensus phosphorylation site mutant PHB2 were expressed as GST fusion protein in BL21 under the induction of IPTG. GST fusion proteins were purified on glutathione-sepharose beads. 5 μl and 10 μl of purified recombinant proteins were separated on SDS-PAGE and stained by coommassie blue. (B) 293T cells were cotransfected with His-CaMKIV along with either CaMKKca or an empty vectors and then His-CaMKIV was pulled down with talon beads and incubated with 10 μl of recombinant wild type or mutant PHB2 in kinase buffer plus [γ-³²P]ATP. Abbreviations: wt, wild type; mut, mutant; ca, constitutively active.