Figure 1.

LPS treatment of ANA-1 cells stimulates binding of Hsp70 and induces expression of CD40. Cells were incubated with LPS or mock-treated as outlined in Materials and methods. (A) Cells were incubated either with biotinylated Hsp70, Hsp70 loaded with biotinylated peptide C, or with biotinylated GST as a control. After 30 min at 4°C, cells were washed, incubated with TRITC-labeled streptavidin, washed again, and processed for fluorescence microscopy. (top) mock-treated ANA-1 cells; (bottom) ANA-1 cells after treatment with LPS. Note that Hsp70 carries on average five biotins, whereas peptide C contains a single biotin. (B) Cells were harvested, lysed, and centrifuged to obtain a total membrane pellet. Identical protein amounts of the samples were analyzed by immunoblotting with an antibody directed against CD40. (Lanes 1 and 4) total cell lysates; (lanes 2 and 5) membrane fractions; (lanes 3 and 6) supernatants.