Figure 4.

Exon 17b–encoded peptides interact with actin. A, schematic diagram of the interacting regions of exon 17b and actin. Number of amino acid residues is indicated. The 50–150 amino acids of exon 17b fused with pGBKT7 was co-transformed with a human kidney library in pGADT7 and analyzed for β-gal activity. Positive actin clones were sequenced, and the region for interacting with exon 17b peptide was identified. B, purified exon 17b peptides cleaved from GST–exon 17b used in gel overlay assays. C, blot overlay assays for the interaction between exon 17b and actin. Actin and BSA (left) or GST−17b and GST (right) are shown by Coomassie Blue staining. A duplicate gel with the same amount of proteins as in the Coomassie Blue–stained gel was separated by SDS-PAGE. Actin and BSA on the membrane were overlaid with exon 17b peptide (left), and GST−17b and GST on the membrane was overlaid with biotinylated actin (right), and followed by Western blotting with an anti-ex17b antibody (left) or horseradish peroxidase-streptavidin (right). D, GST-pulldown analyses for the interaction between exon 17b encoded peptide and actin. GST–exon 17b or GST immobilized on GSH-Sepharose beads were incubated with actin and analyzed for the presence of actin with the beads with an anti-actin antibody. Molecular mass markers (kDa) are provided at the left margin of each blot.