Other methods. The yeast two-hybrid assays were performed as described previously [19]. Briefly,
PaTrin 2 C-terminal 30-kDa domain of Hsc70 and its mutants in pAS2-1 and SL4 in pACT2 were co-transformed into Saccharomyces cerevisiae strain Y190. The β-galactosidase activity of the transformants was determined by filter assays. The pull-down assays using the β-sandwich subdomain of Hsc70 fused with GST was performed by the procedures described in Liu et al. [19]. To purify anti-p49/STRAP antibodies, the antigen first was cross-linked to AminoLink Coupling Gel (Pierce). The antiserum was then incubated with the resin, and the bound
antibodies were eluted with 20 mM HCl. They were immediately neutralized with Tris base. Immunoprecipitation was carried out as described in Liou et al. [26]. The ATP-hydrolytic activity of Hsp70 was measured as previously described [6] and the in vitro refolding assays were performed by the methods described in Wu et al. [21], except that the HDJ1 and Hsp70 used were purchased from Stressgen (Ann Arbor, Michigan).
