File S1 - Limited Promiscuity of HLA-DRB1 Presented Peptides Derived of Blood Coagulation Factor VIII

Figure S1. Subcellular fractionation of immature and mature moDCs on a sucrose density-gradient. Presentation of FVIII was compared between immature DCs and LPS-matured DCs. Cells were homogenized and PNS was fractionated on a sucrose density-gradient. A. Fractions from the sucrose gradient were analyzed by ELISA for MHC class II molecules and divided into 3 pools per sample as indicated. B. Peptides from each pool were identified by mass spectrometry after tryptic digestion. Identified proteins were annotated based on subcellular localization or function using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resource. C. Accession numbers of proteins identified in the different subcellular fractions. Figure S2, Presentation of FVIII peptides by moDCs and macrophages. A. Intensity plot showing reproducibly detected peptide ions across duplicate analyses. Total cell lysates from FVIII-treated moDCs or macrophages derived from donor 2 were used to purify HLA-DRB1-presented peptides using an anti-MHC class II antibody. SIEVE was used to compare intensities of individual peptides and average intensities of each identified peptide are plotted. The diagonal line indicates an equal intensity under each condition and the dotted lines indicate 2-fold differences in intensity. B. FVIII peptides identified in this experiment are listed with sequence, average intensity in cell lysate from macrophages, dendritic cells and intensity ratios between those two conditions. (PDF)