Association of Hsc70 with p49/STRAP in vitro. (A) GST pull-down assays: purified GST (lane 1) or GST fused with the β-sandwich region (lane 2) were incubated with p49/STRAP (lane 3), and the mixtures then were reacted with glutathione-Sepharose. The material bound was eluted with 50 mM of glutathione and was subjected to gel electrophoresis. Coomassie-stained gels are shown here. While p49/STRAP did not associate with GST (lane 4), it
Dactolisib was pulled down by the GST fusion protein (lane 5). The location of p49/STRAP on the gels is indicated by the arrow. (B) Immunoprecipitation: His-tagged Hsc70 [19] was purified (lane 1) and was incubated with p49/STRAP (lane 2) in the presence of ATPγS or ADP. Then, the proteins were precipitated with anti-p49/STRAP
antibodies previously cross-linked with Protein A-Sepharose, and the protein bound was analyzed by gel electrophoresis. A similar, if not identical, amount of Hsc70 was precipitated with p49/STRAP in ATPγS (lane 3) and in ADP (lane 4). Without p49/STRAP, no Hsc70 was brought down by the resin (lane 5). Molecular weight markers were from Invitrogen (pre-stained), and were calibrated using phosphorylase b (97,000), BSA (66,000), ovalbumin (45,000) and carbonic anhydrase (31,000).
