FIGURE 3:

(A) H295R cells were transfected with WT or T759A-mutant pEGFP-DIAPH1 and treated for 30 min with 0.4 mM Bt₂cAMP, and lysates were immunoprecipitated using an anti-GFP antibody and protein A/G agarose. Immobilized proteins were washed, separated by SDS–PAGE, and analyzed by Western blotting using an anti–phospho(T759)-DIAPH1 antibody. Five percent of inputs were subjected to SDS–PAGE and Western blotting using an anti-GFP antibody. Left, representative blots; right, densitometric analysis. Graph data represent mean ± SD of three experiments, each in duplicate. (B) H295R cells were pretreated with 10 μM H89 or 10 μM U0126 and then treated with 0.4 mM Bt₂cAMP for 30 min. Cell lysates were harvested and separated by SDS–PAGE, followed by Western blotting using anti–phospho(T769)-DIAPH1or anti-DIAPH1 antibodies. Left, representative blots; right, densitometric analysis. Graph data represent the mean ± SD of five experiments, each in duplicate. (C) H295R cells were pretreated with H89 or U0126 and then treated with Bt₂cAMP. Cell lysates were harvested and separated by SDS–PAGE, followed by Western blotting using anti–phospho-ERK1/2 or anti-ERK2 antibodies. Left, representative blots; right, densitometric analysis. Graph data represent mean ± SD of five experiments, each in duplicate. (D) Lysates isolated from control or Bt₂cAMP-treated cells were incubated with λ-phosphatase and then subjected to SDS–PAGE and Western blotting for pDIAPH1 or DIAPH1. Left, representative blots; right, densitometric analysis. Graph data represent the mean ± SD of two experiments, each in duplicate. In all graphs, asterisks denote statistical significance (p < 0.05) compared with untreated control.