Figure 3

Tyrosine phosphorylation in the activation loop of HIPK1, HIPK2 and HIPK3. HeLa cells were transfected with expression plasmids for wild type GFP-HIPK fusion proteins or the Tyr→Phe mutants thereof (YF). If indicated, cells were treated with sodium orthovanadate (Na₃VO₄) for 1 h or 2 h. Western blots of total cell lysates were detected with antibodies directed against pTyr361 in HIPK2, a general antibody for phosphotyrosine independent of the sequence context (PY99) and a GFP antibody. A, The pTyr361(HIPK2) antibody detects wild type HIPK1-3 but not the Tyr→Phe mutants. B, Effect of vanadate treatment. The column diagrams show the quantitative evaluation of 3 experiments. The background signal in the untransfected control samples was subtracted from the signal intensities obtained with the phosphospecific antibodies. Relative phosphorylation after vanadate treatment was calculated by normalization to the signal measured in untreated cells. Means + SEM, * p < 0.05, analysed by one-sample t test.