Figure 1
From: The Hsp90 machinery facilitates the transport of diphtheria toxin into human cells
In vitro, the enzymatic domain of DT (DTA) specifically binds to the purified recombinant proteins Hsp90, Hsp70, Hsc70, CypA, Cyp40, FKBP51, and FKBP52 and the isolated PPIase domains of FKBP51 and FKBP52. (a) Dot blot analysis of the interaction between DTA and recombinant chaperones and PPIases. A serial dilution (2 µg, 1 µg, 500 ng and 250 ng) of each of the indicated recombinant proteins was immobilized on a nitrocellulose membrane by vacuum aspiration with the dot blot system and the transfer confirmed by protein staining with Ponceau S (Supplementary Fig. 1). After blocking of the membrane with 5% non-fat dry milk in PBST, the membrane was cut into two identical portions. With one portion, an overlay with biotinylated DTA in PBST (B-DTA, 9.5 nM) was performed for 1 h. After washing, the bound biotin-DTA was detected with streptavidin-peroxidase and the ECL system. For control, the other portion was incubated with PBST instead of B-DTA and then treated exactly as the first portion. The direct binding of DTA to the PPIase domains of FKBP51 (FKBP51FK1) and FKBP52 (FKBP52FK1) was tested exactly as described before. (b) Dot blot analysis of the binding of denatured and native DTA to immobilized chaperones and PPIases. The comparable experiment as described in A was performed with denatured B-DTA in direct comparison to native B-DTA in the overlay. Therefore, DTA was incubated for 1 h at RT either with PBS (native B-DTA) or with 6 M guanidine hydrochloride to obtain denatured B-DTA. (c) Co-precipitation of His6-DTA with Hsp90, Hsp70 and Cyp40 from cell lysate. HeLa lysate (1,500 µg) was incubated for 30 min at RT with His6-DTA (4 µg and 8 µg) or without DTA for control. The subsequent pull-down of His6-DTA was performed by incubation with Talon® CellThru beads for 1 h at 4 °C. After extensive washing of beads, the samples were boiled with SDS-sample buffer, followed by Western blot analysis of co-precipitated proteins with specific antibodies against Hsp90, Hsp70 and Cyp40. The blot membranes were cut prior to antibody incubation to detect the respective proteins. Equal protein loading was confirmed by input control which was taken from the respective sample prior to bead incubation.
