FIG. 3.

Effects of Hap46 and Hop/p60 (Hop) on substrate binding to hsc70. ³⁵S-labeled ER was used as the substrate. It was synthesized in vitro with reticulocyte lysate and subsequently denatured by treatment with urea (see Materials and Methods). Following dilution, the material containing ∼0.3 μM hsc70 (endogenous to reticulocyte lysate) was used without purification. For incubations, Hop/p60 or GST fusion proteins of Hap46 or Hap46(63-274) were added (3 μM each). hsc70-specific immunoprecipitation with K-19 antiserum was done as described in Materials and Methods. Retained material from identical incubations was analyzed either for ER by SDS-PAGE and autoradiography (upper panel) or for Hop/p60 by immunoblotting with antibody F5 (lower panel). In controls without antibody, there were no significant amounts of ER or Hop/p60 (data not shown).