FIGURE 6.

Hsp83 physically interacts with Met/Gce and facilitates Met to bind to JH. A, B, and D, Kc cells were co-transfected with the pAC5.1 expression constructs of Myc-Hsp83 and Met-V5 or Flag-Gce for 48 h, pretreated with 1 μg/ml of GA for 2 h and treated with 1 μm methoprene (JHA) or DMSO for 10 min as indicated, and then cell lysates were subjected to immunoprecipitation (IP) with an anti-Myc (A and D) and an anti-V5 (B) antibody, respectively. The interacting proteins were detected on immunoblot (IB). Input panels represent 10% of the initial material. C, left panel: the bHLH, PAS-A, and PAS-B domains of Met (H*, A*, and B*) were mutated (partially deleted) individually. Right panel: besides the WT (wild type), the mutated Met-V5 constructs were used, the other conditions are the same as in A. E, GA decreased the JH binding activity of Met. Kc cells were transfected with the pAC5.1 expression constructs of EGFP or Met-V5 for 48 h, pretreated with 1 μg/ml of GA for 2 h, the JH-binding activity of total proteins isolated from Kc cells was measured. EGFP, enhanced green fluorescent protein.