Epitope mapping of monoclonal antibody R2-25. (A) Schematic diagram of truncation constructs of Hsc70. A linear diagram of full-length Hsc70 is shown at the top; numbers indicate the corresponding amino acid position. The ATPase domain (ATPase), peptide binding domain (PBD), and C-terminal region (C) are shown. The truncation constructs generated are illustrated below and aligned corresponding to the full-length protein. The name of each construct is listed to the left of the illustration and all have an N-terminal trigger factor (TF). (B) Immunoblot analysis of Hsc70 truncation constructs. Purified proteins separated by SDS-PAGE and stained with Coomassie brilliant blue (CBB) (left panel), and the same samples were analyzed by immunoblotting with R2-25 antibody. TF, trigger factor; C, loading buffer. (C) Sequence of Hsc70 from 385 to 646 amino acids. Six synthesized peptides are shown underlined (PBD-1 to -5, and C-1). (D) Reaction activity of each synthetic peptide was measured by ELISA. Statistically significant differences between control (BSA) and C-1 (EEVD) peptides are indicated by asterisks (*, P < 0.01).