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. 2002 Sep 30;158(7):1277–1285. doi: 10.1083/jcb.200208083

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Copyright © 2002, The Rockefeller University Press

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Figure 3. — Binding of Hsp70 to CD40 is direct and depends on ADP. (A) HeLa cell lysates were incubated with GST (control) or GST-CD40 in the amounts indicated. Thereafter, samples were affinity-purified on glutathione-sepharose as outlined in Materials and methods, subjected to SDS-PAGE, and analyzed by immunoblotting with antibodies directed against Hsc70 and Hsp70 (top lanes), and against Hsp90 (bottom lanes). (B) Human recombinant His6-tagged Hsp70 was incubated with ATP, ADP, or an excess of peptide C, followed by addition of CD40-GST or GST alone. After affinity purification, samples were analyzed by immunoblotting as described in A, using antibodies directed against the His6 tag. (C) Recombinant human His6-tagged Hsp70 was incubated with peptide C, either in the presence or absence of ADP. The reactions were then incubated with ANA-1 cell lysates as described in Materials and methods. Protein bound to Hsp70 was analyzed by affinity purification on Ni-NTA agarose and immunoblotting with antibodies directed against CD40. Input reflects the amount of protein subjected to affinity purification.