FIGURE 1.

IMPDHII is recruited to lipid rafts following TLR2 stimulation and is subjected to post-translational modifications. A, left panel, image of one gel selected from the 12 gels realized for two-dimensional DIGE analysis. This gel contains lipid rafts proteins prior to (Cy3, green) and following a 5-min stimulation with Pam3 (Cy5, red). Cy2 is an internal standard and corresponds to a 1:1 ratio of unlabeled proteins from both groups. Right panel, DeCyder relievo presentation of the identified IMPDHII spot volume in control, Pam2-, and Pam3-stimulated THP1 cells. Gels were analyzed by DeCyder software, and statistics were generated for up-regulated and down-regulated proteins such as IMPDHII. B, confirmation of two-dimensional DIGE findings by immunoblotting. THP1 cells were stimulated with Pam2 or Pam3 (100 ng/ml) and lysed with 1% Triton X-100. Lipid rafts were isolated by ultracentrifugation (fractions 2–4), and lipid raft proteins were subjected to SDS-PAGE. IMPDHII was revealed by immunoblot in lipid rafts. C, lipid rafts were isolated from THP1 cells stimulated with Pam3 (100 ng/ml) for 5 min and subjected to two-dimensional electrophoresis. Two-dimensional gels were then transferred onto nitrocellulose membranes and subjected to immunoblot with anti-IMPDHII antibodies. D and E, phosphorylation changes following stimulation of TLR2 by lipopeptides. D, lipid raft phosphoprotein profiles of nonstimulated and stimulated (Pam3, 5 min) THP1 were viewed on a two-dimensional gel using in-gel staining with ProQ Diamond. Total protein stain of lipid rafts was performed with SYPRO Ruby. Here is a “zoom” view of a two-dimensional gel, centered on one spot identified by MALDI-TOF as IMPDHII (circles). E, THP1 cells were stimulated with Pam3, and cell lysates were immunoprecipitated with 3 μg of IMPDHII monoclonal antibodies. Tyrosine-phosphorylated proteins were then visualized by immunoblot with anti-phosphotyrosine antibodies (4G10 clone). IB, immunoblot; NS, nonstimulated.