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. Author manuscript; available in PMC: 2014 Jan 15.
Published in final edited form as: J Immunol. 2012 Dec 12;190(2):723–736. doi: 10.4049/jimmunol.1201646

FIGURE 1.

FIGURE 1

CD86 associates with Phb1 and Phb2 in B Lymphocytes. A, CH.12LX B cells were primed with CD40L/IL-4 for 16 hours followed by the addition of a species- and isotype-matched control Ab (anti-rat IgG2a ctrl Ab), or rat anti-mouse CD86 (GL1) (anti-CD86) for 15 minutes. Lysates were prepared, followed by immunoprecipitation (Pierce). Immunoprecipitates were separated via SDS-PAGE followed by total protein staining with Sypro Ruby. Unique bands in the CD86 immunoprecipitate are indicated by arrows at ~50, ~35, and ~28 kDa. B, The band marked with an arrow at ~35 kDa was sequenced via LC/MS Mass Spectrometry. Tryptic peptide fragments were sequenced and matched with known tryptic peptides via a MASCOT search. 14 non-redundant tryptic peptides were identical to murine prohibitin-2 (Phb2). C, CH12.LX B cells alone, CH12.LX B cells expressing a FLAG-CD86 recombinant protein, or primary splenic B cells were primed as described above. Immunoprecipitates were immunoblotted for CD86, Phb2, and Phb1. Gels are representative of three independent experiments. D, FLAG-CD86 transfected CH12.LX B cells with either Full Length FLAG-CD86 or a Truncated FLAG-CD86 (KKPΔ) or non-transfected B cells from WT or Trunc CD86-expressing mice were primed as described above, immunoprecipitated with either anti-FLAG or anti-CD86, and were analyzed for Phb1/2 and CD86 protein levels via immunoblot. The gels shown are representative of three independent experiments (left panel) or one experiment (right panel).