Identification of CD85j ligand(s) expressed by HIV-1-infected MDDC. (A) MDDC were lysed at day 2 post HIV-1 infection. After blocking CD85j receptors and HLA class-I ligands as described in Materials and Methods, CD85j-Fc fusion protein was used to capture and purify proteins from uninfected or HIV-1-infected MDDC lysates onto the Protein chip array. Eluted proteins from the column were separated by electrophoresis in non-reducing conditions and revealed by silver staining. Lane 1, Seeblue® Plus2 Pre-Stained Standard, lanes 2–7, subsequent eluted fractions of HIV-1-infected MDDC lysates after pre-incubation with the CD85j-Fc-coupled gel from two representative donors (lanes 2–4, donor 1; lanes 5–7, donor 2). The proteins were identified as belonging to S100A8/A9 complex (S100A8, S100A9 and S100A8/A9) by protein sequencing. (B) Eluted fractions of HIV-1-infected MDDC lysates were analyzed by immunoblotting using anti-S100A9 (lane 1), anti-S100A8 (lane 2) and anti-CD85j (lane 3) mAbs. (C) CD85j-Fc fusion protein was used to capture and purify proteins from uninfected or HIV-1-infected MDDC lysates, where HLA-I molecules were blocked, onto the Protein chip Array.