Figure 5.

FHL1 forms a complex with NFATc1. (A) GST-FHL1, GST-GATA-2, or GST and His-NFATc1 or His were coexpressed as indicated in E. coli. GST proteins and associated proteins were bound to glutathione-Sepharose. Western blots are representative of three experiments. Arrows indicate GST- and His-tagged proteins. The His tag alone did not copurify with GST-FHL1 (not depicted). (B) GST-FHL1 or GST coupled to glutathione-Sepharose was incubated with skeletal muscle lysates from wild-type FVB/n mice and immunoblotted for endogenous NFATc1, GST, or GST-FHL1 as indicated. Results shown are representative of four similar experiments. (C) Coimmunoprecipitation of HA-FHL1 and myc-NFATc1 in C2C12 cells. C2C12 cells were transfected with the indicated constructs and immunoprecipitated with a myc antibody, then immunoblotted with an HA antibody (top). Immunoprecipitation of myc-NFATc1 was confirmed by immunoblotting with myc antibody (middle). Lysates were immunoblotted with an HA antibody to confirm equal expression of FHL1 constructs (bottom). Immunoblots are representative of three experiments. (D) Densitometric quantification of HA-FHL1 wild-type or C132F/H123Y mutants, relative to immunoprecipitated myc-NFAT. The bar graph represents the mean ± SEM (n = 3 experiments; **, P < 0.01).