Figure 1. Peptide P140 binds the constitutively-expressed chaperone HSC70 protein (A) Biotin-labeled P140 and 131-151 peptides form a stable and specific, dose-dependent complex with HSC70 expressed on the surface of CBA/J and MRL/lpr splenocytes.

The same results were obtained with LNCs from normal and lupus mice. There is no binding when a biotin-labeled scrambled form of P140 peptide (ScP140 Btn) is used instead of P140 peptide, and the binding is inhibited by an excess of free, non-biotinylated peptides (see the quantification by densitometry). (B) Direct binding to recombinant HSC70 protein of P140 and non-phosphorylated 131–151 peptides used at increasing concentrations (1.56 to 12.5 µM), as measured by surface plasmon resonance experiments. Analysis was performed using the simple 1∶1 Langmuir binding model, the fitting to each model was judged by the χ² value and randomness of residue distribution was compared to the theoretical model. (C) Subcellular localization of HSC70 shown by immunoelectron microscopy. Fixed and permeabilized MRL/lpr PBLs were incubated overnight with the anti-HSC70 rat monoclonal antibody 1B5, followed by incubation with goat anti-rat IgG conjugated to ultra-small gold particles. Gold particles were enhanced using a silver kit. Cells were then treated for transmission electron microscopy with conventional methods (bar = 500 nm). (D) Co-localization, at the surface of non-permeabilized MRL/lpr splenocytes, of P140 peptide and HSC70 staining as shown by fluorescent microscopy: P140 followed using biotinylated peptide and FITC-labeled streptavidin, and HSC70 followed using the R-phycoerythrin-labeled anti-HSC70 rat monoclonal antibody 1B5. DAPI (4′,6′-diamidino-2-phenylindole) was used to label the nucleus (bar = 3 µm). (E) Binding in a dose-dependent manner of biotin-labeled P140 peptide (but not of ScP140 Btn) to the surface of MRL/lpr splenocytes as measured at 4°C by FACS analysis (the results obtained at 20 and 37°C are shown in Figure S4). (F) Cell-surface HSC70 expression among different CBA/J (black line) and MRL/lpr (red) cell subsets. (1) B220⁺TCRβ⁻ = B cells; (2) B220⁺TCRβ⁺ = activated T cells frequent in lupus mice, including CD4⁻CD8⁻ (DN) T cells, the number of which is raised in MRL/lpr mice; (3) B220⁻TCRβ⁻ = naive cells; (4) B220⁻TCRβ⁺ = T cells. Binding visualized with an isotypic control antibody is shown (right panel, in green).