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. 2014 Apr 8;53(16):2701–2709. doi: 10.1021/bi5001728

Figure 6.

Figure 6

Interaction of MYPT1 with myosin. (A) Structure of myosin II heavy chain. Abbreviations: S-1, subfragment-1; S-2, subfragment-2; LMM-N, N-terminal portion of light meromyosin; ACD, assembly competent domain. (B and C) Binding of recombinant MLCP to S-tagged myosin fragments. S-tagged S-2, LMM-N, and ACD were expressed in COS1 cells and captured with S-protein affinity beads. The beads were mixed with cell lysates, including the complex of myc-MYPT1 with 3×HA-PP1C (B) or myc-MYPT1(1–498) with 3×HA-PP1δ and myc-MYPT1(495–1030) with 3×HA-M20 (C). The proteins bound to the beads were analyzed by immunoblotting. (D) Subfractionation of cells expressing recombinant MLCP. myc-MYPT1(1–498) (“N”) and myc-MYPT1(495–1030) (“C”) were co-expressed in COS1 cells with 3×HA-PP1C (H124N) and 3×HA-M20, respectively. Cells were lysed with buffer [10 mM HEPES (pH 7.4) with 0.2 M NaCl, 5 mM Mg(OAc)2, 2 mM EGTA, and 0.1% Triton X-100] and subjected to airfuge for 30 min at 80000 rpm. S and P denote the cytoplasm and cytoskeletal fractions, respectively.