(A–C) Cardiomyocytes were uninfected (Un), infected with empty virus (EV) for expression of FLAG alone, or infected with adenoviruses for expression of wild-type (WT) MAP4K4 (M4K4). Cells were treated with calyculin A (CalA; 200 nM, 10 min) or DMSO (1/2000 dilution) and extracts were prepared. Proteins were immunoprecipitated using anti-FLAG antibodies, and equal volumes of the total extract, immunoprecipitated proteins (IP) and supernatants (SN) were immunoblotted with antibodies to MAP4K4, Strn, Strn3 or Strn4. The concentration of proteins in the IP relative to the SN for the same cell extracts is indicated. (A) Data for two independent cardiomyocyte preparations (1 and 2) are shown. (B) One of two similar experiments is shown. (C) Densitometric analysis of the blots in A and B to show the percentage of each striatin associating with WT-MAP4K4. The raw densitometric values were adjusted for final volumes of the SN and the IP, and the percentage in the IP calculated relative to the total amount (SN + IP). (D,E) Cardiomyocytes were uninfected, or infected with adenoviruses containing empty vector or constructs for WT-MAP4K4 (WT-M4K4), MAP4K4(T187A) [M4K4(A)] or MAP4K4(T187D) [M4K4(D)]. Cells were treated with H₂O₂ (1 mM, 15 min; D) or TNFα (25 ng/ml, 15 min; E) and extracts (25 µg) immunoblotted with antibodies for phosphorylated (phospho-) or total JNKs, or the FLAG epitope. Representative blots are on the left (positions of relative molecular mass markers are indicated on the right of each blot) with densitometric analysis on the right. Results are means ± S.E.M (n = 5 independent cardiomyocyte preparations). Individual P values are given (repeated measures two-way ANOVA with Holm–Sidak post-test).