AdV-infected myocytes expressing (A–C) FLAG–MST3(FL) or (D) FLAG–MST3(T328A) were unstimulated or exposed to calyculin A (100 nM for 10 min) and the extracts were subjected to gel-filtration chromatography using Sephacryl S300HR. (A–D) Column fractions were immunoblotted with anti-FLAG antibodies: (A) fractions from unstimulated myocytes; (B) fractions from myocytes exposed to calyculin A; (C) fractions from mixed extracts from unstimulated myocytes and myocytes exposed to calyculin A; and (D) fractions from unstimulated myocytes (left-hand panel) or myocytes exposed to calyculin A (right-hand panel). In (A), M is a marker lane of FLAG–MST3(FL) in whole myocyte extracts. In (C), M is a marker lane of mixed whole extracts of FLAG–MST3(FL) from myocytes unexposed or exposed to calyculin A. (E) Elution profiles for the quantified imunoblots. ●, FLAG–MST3(FL); ■, FLAG–MST3(FL) from myocytes exposed to calyculin A; and ▲, FLAG–MST3(T328A). (F) The Sephacryl S300HR column was standardized with proteins of known Stokes radii (taken from the literature). 1: ferritin, 61.0 Å; 2: catalase tetramer, 52.0 Å; 3: IgG, 50.0 Å; 4: BSA dimer, 47.5 Å; 5: catalase dimer, 45.0 Å; 6: BSA monomer, 35.0 Å; 7: Hb, 31.0 Å; 8: cytochrome c, 17.0 Å. Experiments were repeated at least twice with different myocyte preparations with similar results.