FIGURE 4.

SBD and CTD domains of Hsc70 interact with TH. A, schematic of Hsc70 and GST fusion proteins containing three functional domains: the nuclear binding domain (ATPase, residues 1–373), SBD (residues 373–540), and CBD (residues 540–650). B, even amounts of immobilized GST fusion proteins were used for pulldown experiments in MN9D cell and rat brain lysates. Samples were analyzed by SDS-PAGE and IB with antibodies against TH (first panel) and AADC (second panel). As a negative control, the pulldown samples were also analyzed using the unrelated GS antibody (third panel). Ponceau red staining (fourth panel) shows even loading of the fusion proteins. C, in vitro binding assays were performed by incubating purified His₆-TH with even amounts of the Hsc70 GST fusion proteins or GST-only prior to SDS-PAGE and IB analysis using anti-TH antibody.