Figure 3.

Co-immunoprecipitation (Co-IP) of H. capsulatum peptides linked to MHC-II from BMDM and BMDC. (A) Schematic representation of Co-IP. (I and II) Mφ and DCs were differentiated from bone marrow cells extracted from C57BL/6 mice. (III) After 4 hours of co-culture of murine cells with heat-killed yeast cells, (IV) MHC-II extraction was performed. (V and VI) Magnetic beads containing G protein were complexed with anti-mouse IgG2a I-Ab. After the incubation period, the amount of antibody in the supernatant was measured and total binding of antibody to magnetic beads was achieved. (VII) The extract was incubated for affinity capture of the MHC-II + peptide complexes. (VII) Then the peptides were dissociated from the column after acid denaturation and (IX) the eluted peptides were identified by nanoLC-MS/MS. (B) 1-D SDS-PAGE of the eluted peptide sample. Electrophoresis gels were stained with silver nitrate. kDa: kilodaltons. (C) Venn diagram showing common and exclusive proteins obtained through 2-D gel electrophoresis and Co-IP of H. capsulatum peptides linked to MHC II from BMDM and BMDCs. Orange ellipse: Proteins presented by BMDM. Green ellipse: Proteins presented by DMDC. Blue ellipse: proteins sequenced from 2-D gel extraction. Crossing the ellipses represents the proteins that were identified in common among Co-IP (BMDM and BMDCs) and 2-D gel electrophoresis. (D) Venn diagram showing common and exclusive peptides obtained Co-IP of H. capsulatum peptides linked to MHC II from BMDM and BMDCs. The results represent peptides sequenced by nanoLC-MS/MS. Orange ellipse: peptides presented by BMDM. Green ellipse: peptides presented by DMDC. Crossing the ellipses represents the peptides that were identified in common among BMDM and BMDCs.