Fig. 4.

Two-distinct binding sites reside within the amino-terminus of Int. The chitin-binding domain (CBD) and the CBD–Int amino-terminal fusions (CBD–Int 1–62 and CBD–Int 1–75) were used to affinity precipitate full-length Int or C75, an amino-terminus-truncated Int as described in Experimental procedures. Briefly, reaction mixtures containing the appropriate Int protein, full-length wild-type or C75, were incubated with either the CBD alone or a CBD–Int fusion. The reactions were incubated at 4°C for 2.5 h and the CBD complexes precipitated via the addition of chitin. The precipitates were subsequently washed and loaded onto denaturing 12% SDS-PAGE gels. The proteins in the gel were subsequently transferred to PVDF membrane and analysed via Western blotting with (A) polyclonal rabbit anti-Int and (B) polyclonal rabbit antichitin binding domain antibodies. Results are representative of at least three independent experiments.