FIGURE 5.

A, anti-CypA immunoblot of sucrose density fractions of conditioned medium from high density clone C cells. B, cellulose-bound hensin 12-mer peptides on a SPOT array were incubated with CypA either in the absence of CsA (left) or in the presence of CsA (right), transferred to nitrocellulose membranes, and immunoblotted with anti-CypA antibody. Yellow and green bars indicate the peptide regions of hensin that bound to CypA. Peptides in which interaction with CypA could not be competed off by preincubation with CsA are underlined in green, and those successfully competed off by preincubation with CsA (underlined in yellow) are thereby possible specific binding sites for the PPIase active site of CypA. See supplemental Fig. 2 for detailed sequence information of these peptides. C, conditioned medium was added to Ni-NTA beads coupled with recombinant CypA in the presence or absence of CsA and to the appropriate control beads. Bound protein was eluted off from the beads using imidazole elution buffer twice (elution 1 (lane E1) and elution 2 (lane E2)) and immunoblotted with anti-hensin antibody.