Fig 10.

 Association of recombinant Hsp30C (30C) and mutants with luciferase (LUC) and Hsc70 during refolding. (A) Association of 30C with LUC during refolding. LUC (0.2 μM) was maintained at 22°C for 15 minutes with 6 μM 30C, and then incubated with reticulocyte lysate (RRL) at 30°C for either 0 minute (lane 1) or 40 minutes (lane 2). Alternatively, LUC was heat denatured at 42°C with 30C, and then incubated with RRL for 0 minute, 40 minutes, 60 minutes, or 90 minutes (lanes 3–6). LUC and associated complexes were immunoprecipitated using an anti-LUC antibody and immunoblot analysis with an anti-30C antibody. (B) Comparison of 30C, N-30C, and C-30C association with LUC during refolding. LUC (0.2 μM) was heat denatured with 6 μM 30C (lanes 1 and 4), N-30C (lanes 2 and 5), or C-30C (lanes 3 and 6). Samples were then incubated with RRL at 30°C for either 0 minute (lane 1–3) or 40 minutes (lanes 4–6). LUC and associated complexes were immunoprecipitated using an anti-LUC antibody and immunoblot analysis with an anti-30C antibody. (C) LUC heat denatured with 30C associates with Hsc70 during refolding. Hsp30C (6 μM) was incubated alone at either 22°C (lane 1) or 42°C (lane 2), or with LUC (0.2 μM) at 22°C (lane 3) or at 42°C (lane 4) for 15 minutes. LUC incubated alone at 42°C is shown in lane 5. The samples were combined with RRL and adenosine triphosphate and kept at 30°C for 20 minutes. Samples were subjected to immunoprecipitation using rabbit polyclonal anti-30C and immunoblot analysis with mouse monoclonal anti-Hsp70 antibody