Myocytes were uninfected (A), infected with AdV–FLAG–MST3(FL) (B–F and H) or infected with AdV–GST–MST3(FL) (G). They were exposed to 100 nM calyculin A for the times indicated and extracts were prepared. Proteins were separated by SDS/PAGE (A–C) or (E–G) phos-tag™ SDS/PAGE (D and H) followed by immunoblot (IB) analysis. (A) Extracts were immunoblotted with anti-MST3(275–393) mAbs and the bands (left-hand panel) were quantified (right-hand panel). (B) Extracts were immunoblotted with anti-MST3(275–393) mAbs (upper panel) to detect endogenous MST3 and FLAG–MST3(FL) or with anti–FLAG antibodies (Abs, lower panel) to detect FLAG-MST3(FL). (C) Myocytes were unstimulated, or exposed to calyculin A in the absence or presence of staurosporine (1 μM) and extracts were immunoblotted with anti-FLAG antibodies. (D) Extracts were analysed using phos-tag™ SDS/PAGE (left-hand panel) or SDS/PAGE (right-hand panel) and immunoblotted with anti-FLAG antibodies. (E–H) Myocytes expressing FLAG–MST3(FL) (E, F and H) or GST–MST3(FL) (G) were unstimulated or exposed to calyculin A for 10 min. FLAG–MST3(FL) was immunoprecipitated using anti-FLAG M2 affinity gel and GST–MST3(FL) was precipitated using glutathione–Sepharose. Precipitates were incubated with PP2A for the times indicated. In some instances (F and H), 2 μM okadaic acid was included in the PP2A incubations. Extracts were immunoblotted with anti-FLAG (E, F and H), anti–MST3(275–393) mAbs (G, upper panel), or anti-GST antibodies (G, lower panel). (E) The lanes showing extracts of myocytes exposed to calyculin A (upper panel) were quantified (lower panel). ●, upper band; ■, lower band. Experiments were repeated at least twice with similar results with different myocyte preparations.