Fig. 1. (A) Regions of N-RAP expressed as GFP fusion proteins. GFP was fused to the N terminus of full-length N-RAP (1), the N-RAP super repeats (N-RAP-SR) (2), the region in between the super repeats and the LIM domain (N-RAP-IB) (3), and the N-RAP LIM domain (N-RAP-LIM) (4). Numbers above the diagram refer to amino acid residues from the full-length mouse N-RAP sequence, while Ab marks the position of a 30 residue peptide used as an antigen for the production of polyclonal antibodies. The predicted molecular weights of the N-RAP fusion proteins are indicated in parentheses, including the 28.6 kDa contributed by the N-terminal GFP. (B) Immunoblot analysis of chick cardiomyocytes transfected with GFP-N-RAP-SR (lane 1), GFP-N-RAP-IB (lane 2), GFP-N-RAP-LIM (lane 3) and GFP alone (lane 4). Equivalent volumes of lysate from cultured chick cardiomyocytes were loaded in each lane and probed with anti-GFP antibody. In each case, the detected band migrated near the position predicted from the sequence of the fusion plasmid. The detected bands are labeled (right), together with the position of the molecular weight markers (left). Note that GFP alone contains 4.7 kDa attributed to 46 C-terminal residues that are not present in the N-RAP fusion proteins, owing to the stop codons engineered into the fusion constructs at the precise end of the sequences derived from N-RAP (Table 1).
