Fig. 1. (A) Schematic diagram of N-RAP domain organization (Mohiddin et al., 2003) and regions of N-RAP expressed as GFP fusion proteins. An alternatively spliced single module that is skeletal muscle-specific is shown as a filled box. The position of a 30-residue peptide used as an antigen for the production of polyclonal antibodies is marked as `ab'. Previously described single region constructs contained GFP fused to the N terminus of the LIM, IB and SR regions. The new double region constructs contain each combination of the single regions; these include the LIM + IB construct (LIM-IB), the IB + SR construct (IB-SR) and the LIM + SR construct (LIM-SR). (B) Immunoblot analysis of chick cardiomyocytes transfected with constructs encoding GFP tagged N-RAP constructs. The cells were expressing unfused GFP (lane 1), cardiac IB (lane 2), cardiac IB-SR (lane 3), cardiac LIM-IB (lane 4), LIM-SR (lane 5) and skeletal muscle IB (lane 6). Equivalent volumes of lysate from cultured chick cardiomyocytes were loaded in each lane and probed with anti-GFP or anti-N-RAP antibodies, as indicated. In each case a band was detected migrating near the size predicted from the sequence of the fusion plasmid, which is indicated below each lane. A prominent extraneous band was sometimes also detected at 65 kDa, but did not interfere with the identification of the intact fusion proteins.