Activation of CD44-signaling complex by RANTES. HeLa-CD4 cells (A-D) or primary CD4⁺ T cells (E) were treated (+) for 30 minutes (A-B,D), 15 minutes (C), or 24 hours (E) with 640 nM RANTES or mock treated (–). Then WCEs were immunoprecipitated with indicated antibodies and equivalent amounts of immunoprecipitated samples separated on 4% to 20% (A) or 10% (B-E) SDS-PAGE gels and immunoblotted using indicated antibodies. Molecular weight is indicated on the left in kilodaltons. Asterisk indicates the immunoglobulin heavy and light chains. (A) Serine/threonine phosphorylation after RANTES treatment was investigated in IPs with mouse anti–P-ser/thr mAbs. Total and CD44-specific serine/threonine phosphorylation was detected by immunoblotting with mouse anti–P-ser/thr mAbs (left panel) and anti-CD44 mAb clone F10-44-2 (right panel). Protein bands with changes in phosphorylation state are indicated by roman numerals. (B) Interaction of CD44 with tyrosine-phosphorylated proteins was analyzed in CD44 IPs by immunoblotting using mAbs 4G10 (left panel). Protein bands with changes in phosphorylation state are indicated by arabic numerals. The right panel confirms the equal loading of samples by reprobing with anti-CD44 antibodies. (C) Association of CD44 with kinases Fyn and Src was analyzed in CD44 IPs by immunoblotting using rabbit pAbs anti-Fyn and anti-Src mAbs, respectively. (D) Association of CD44 with the focal adhesion kinase p125 FAK and activation of FAK was analyzed in CD44 IPs by immunoblotting using rabbit anti-FAK pAbs and phospho-specific rabbit anti-FAK[pY397] pAbs. (E) Association of CD44 with the adapter molecule Shc was analyzed in CD44 IPs by immunoblotting using rabbit antibody anti-Shc. Isoforms p46 and p66 are indicated. (F) Interaction of CD44 in primary CD4⁺ T cells with tyrosine-phosphorylated proteins was analyzed in CD44 IPs by immunoblotting using mAb 4G10. Protein bands with changes in phosphorylation state are indicated by lowercase letters. One of 2 to 4 individual experiments is shown.