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Fig. 2. Expression of MLCK-GFP fusions in HeLa cells. (A) MLCK truncations. Dark gray boxes represent the Ig motifs, black bars represent the DXR motifs and the light gray box represents GFP. (B) The kinase activities of the immunoprecipitated kinase-dead and wild-type long MLCK-GFP fusions were examined in the presence of Ca²⁺/calmodulin using the free RLC as a substrate. The wild-type long MLCK phosphorylates the RLC in Ca²⁺/calmodulin-dependent manner, because phosphorylation was not detected in the presence of EGTA. The kinase-dead MLCK did not phosphorylate the RLC in the presence of Ca²⁺/calmodulin or Ca²⁺/calmodulin/EGTA. Symbols are as follows: ${bullet}$ , wild-type MLCK in the presence of Ca²⁺/calmodulin; ${circ}$ , wild-type MLCK in the presence of Ca²⁺/calmodulin and 2 mM EGTA; ${blacktriangleup}$ , kinase-dead MLCK in the presence of Ca²⁺/calmodulin; ${square}$ , kinase-dead MLCK in the presence of Ca²⁺/calmodulin and 2 mM EGTA. (C) Immunoblot analysis of HeLa whole cell lysates expressing the GFP-kinase-dead-MLCK and the truncated MLCK-GFP fusions. Lane 1 contains the HeLa kinase-dead long MLCK ( $~$ 237 kDa); lane 2 contains the HeLa IgG domain plus five DXRs ( $~$ 138 kDa); lane 3 contains the HeLa IgG domain ( $~$ 121 kDa); lane 4 contains the rabbit five DXR fragment ( $~$ 45 kDa); lane 5 contains GFP alone ( $~$ 27 kDa). The lower band in lane 4 is caused by proteolysis of the five DXRs. The MLCK IgG domain polyclonal antibodies do not cross-react with the five DXRs. The MLCK monoclonal antibody recognizes an epitope from the third and fourth DXR motifs (Blue et al., 2002), and does not react with the IgG domain. The GFP monoclonal antibody detects all four MLCK-GFP fusions. The endogenous long MLCK ( $~$ 210 kDa) can be detected with both the MLCK monoclonal and MLCK IgG domain antibodies at longer exposure. The bottom panel shows ß-tubulin as a loading control. In addition, immunoblot analysis with antibodies to GFP demonstrated that GFP fusions of the full-length chicken skeletal MyBP-H and MyBP-C proteins are expressed in HeLa cells.

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