Figure 4.

Characterization of I-P58^IPK hsp40 activity. (A) Preincubation of hsp40 with P58^IPK is required for I-P58^IPK activity. The in vitro assay was performed with purified components as described in the legend to Fig. 2A. In the experiments performed without preincubation, GST–P58^IPK and PKR were mixed before the addition of hsp40 to the in vitro reaction. (B) Hsp40 does not induce the degradation of GST–P58^IPK. GST–P58^IPK was either untreated or incubated under the precise in vitro assay conditions with the indicated amounts of hsp40. The reaction was analyzed by SDS/PAGE and Western blot analysis using P58^IPK specific monoclonal antibody, as described in Materials and Methods. The faster-migrating protein is a breakdown product of the GST–P58^IPK fusion protein and does not appear to contribute to the reaction.