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. 2005 May 10;388(Pt 1):17–28. doi: 10.1042/BJ20041236

Figure 2. Calcium-dependent tyrosine phosphorylation of MEKK4.

Figure 2

(A) Effect of calcium chelation by BAPTA/AM and EGTA on MEKK4 tyrosine phosphorylation. HaCaT cells were pre-treated for 15 min with 10 μM BAPTA/AM to chelate intracellular calcium or for 20 min with 3 mM EGTA to deplete extracellular calcium, followed by IFNγ stimulation as indicated. Controls were harvested after chelator pre-treatment. MEKK4 was immunoprecipitated from lysates and immunoblotted (IB) against phosphotyrosine (α P-Y; upper panel). The membrane was stripped and re-probed with polyclonal antibody directed against MEKK4 (α MEKK4; lower panel to show total amounts of protein, as described in the legend to Figure 1(A) (lower panel). (B) Co-immunoprecipitating proteins are affected by depletion of intracellular calcium. Appropriate parts of the same immunoblot were probed with monoclonal antibodies against annexin II (α annexin II; top panel), Pyk2 (α Pyk 2; middle panel) and SHP2 (α SHP2; bottom panel) to visualize the calcium dependency of proteins co-immunoprecipitating with MEKK4. IFNγ stimulation following BAPTA/AM pre-treatment resulted in abolished induction of tyrosine phosphorylation of MEKK4 and eradicated co-immunoprecipitation of annexin II, Pyk2 and SHP2.