Figure 3. Design and in vitro effects of Prep1(54-72) peptide on Prep1/p160 association.

(A) Binding curves of Prep1(59-72) and Prep1(54-72) peptides to p160_20-160 by ELISA-like assays. Biotinylated peptides at several concentrations were added in triplicate to a 96-well plate coated with p160_20-160 (0.1μM). Experiments were performed at least twice and the average results fitted using GraphPad Prism. Fitting of data with a one-site binding model provided a KD value of 0.18μM ± 0.04 and 1.25μM ± 0.08 for Prep1(54-72) and Prep1(59-72) respectively. (B) Competition binding experiments. His6-tagged-p160_20-160 at 0.25μM was incubated with Prep1(59-72) and Prep1(54-72) at concentrations ranging between 0.001÷10μM and added to a 96-well plate coated with Prep1_45-155 (0.10μM). Results are the average of three independent experiments. (C) L6 cells were incubated with increasing concentrations of the fluorescein-conjugated peptide (FITC-Prep1(54-72)) and peptide uptake was detected by FACS analysis of fluorescein-labeled cells. Bars represent the mean ± SD of four independent experiments. (D) L6 muscle cells incubated with Prep1(54-72) were stimulated with C2cer (10μM) for 18h. Protein lysates were immunoprecipitated with Prep1 antibodies and analyzed by Western blot using antibodies for p160 or for Prep1. (E) An aliquot of the lysate was used to measure protein expression of Prep1 and p160 and the beta-actin used as loading control. The blots were detected by ECL and autoradiography. The autoradiograph is representative of three independent experiments. Asterisks denote statistically significant differences (*p<0.05; **p<0.01).