DC binding site or receptor for Hsp70L1. (A) Confocal microscopy. DCs, fixed with paraformaldehyde, were stained with FITC-Hsp70L1, FITC-His-CK, or FITC-Hsp70 at 4°C, then observed by confocal microscope. (B) Internalization of FITC-Hsp70L1 by DCs. Unfixed, viable DCs were allowed to bind FITC-Hsp70L1 at 4°C, then incubated at 37°C with concurrent visualization by confocal microscopy at the indicated times. (C) Competitive binding. Paraformaldehyde-fixed DCs were incubated with FITC-Hsp70L1 and competitor mixtures. FITC-Hsp70L1 was used at a final and fixed concentration of 20 μg/mL. Competitor proteins were used at increasing doses (50-800 μg/mL final concentration), as indicated. Cells were washed extensively to remove unbound protein and analyzed by flow cytometry. Mean fluorescence intensities (MFIs) are plotted as a function of increasing quantities of competitor proteins. Error bars represent 3 independent experiments. (D) Binding assay of Hsp70L1 with TLR2/4. Ni-beads were pre-incubated with the indicated concentrations of purified recombinant His-Hsp70L1 fusion protein, washed, and then incubated with DC cell lysates. Proteins precipitated by Ni-beads were detected by Western blot assay using anti-His, anti-TLR2, or anti-TLR4 antibody. (E) Activation of NF-κB pathway by recombinant Hsp70L1 protein in TLR2- or TLR4-expressing HEK293 cells. HEK293 cells transiently transfected with luciferase reporter (mock, TLR2^–TLR4^–), TLR2/CD14, and luciferase reporter (TLR2), or TLR4/CD14/MD2 and luciferase reporter (TLR4) plasmids were cultured for 24 hours and then treated with 10 μg/mL Hsp70L1, Hsp70, or His-CK for 6 hours. Cells were lysed and NF-κB luciferase activity measured using the Dual-Luciferase Reporter Assay System. Data were normalized by Renilla-luciferase activity and shown as means ± SEM of 3 independent experiments.