Figure 3
Isolation, ubiquitination activity, and structural characterisation of the Hsp70:p53-TMGST:CHIP complex. (a) p53-TMGST ubiquitination assay mediated by Hsp70 and CHIP. For the western blot, an anti-p53 antibody was used. (b) Formation of the Hsp70:p53-TMGST:CHIP complex. Top, size-exclusion profile of the complex (thick continuous line) compared with the profiles for Hsp70 (dotted line), p53-TMGST (thin continuous line), and CHIP (broken line). Bottom, the selected fractions were analysed by SDS-PAGE and stained with Coomassie Blue. (c) Three orthogonal views of the Hsp70:p53-TMGST:CHIP complex 3D reconstruction. (d) The same views with docking of Hsp70 (Hsp70NBD and Hsp70 SBD), p53-TMGST (p53DBD and GST), and CHIP. The DTSSP crosslinks in the images are described in Supplementary Fig. 5c. Only crosslinks between residues located in regions with known atomic structure are depicted in the figure. Colours as in Fig. 2. Bar, 100 Å for (c,d).
