Influence of CD44 expression level on RANTES cell interaction. HeLa-CD4 cells were analyzed 3 days after transfection with CD44e3 dsRNA, invCD44e3 dsRNA, or mock-treated cells for CD44 mRNA and protein expression. (A) CD44 mRNA expression was quantified with real-time RT-PCR. CD44-specific mRNA is depicted relative to mock-transfected control. One of 4 individual experiments is shown. (B) CD44 protein expression was monitored by FACS analysis. The same culture as analyzed in panel A is shown. One of 15 individual experiments is shown. Percentages of cells with low and high CD44 expression levels are indicated. (C) Three days after transfection with CD44e3 dsRNA, invCD44e3 dsRNA, or mock transfection, HeLa-CD4 cells were treated for 24 hours with 640 nM RANTES. CD44 expression and RANTES binding to cells was then monitored by FACS analysis using PE-labeled anti-CD44 and anti-RANTES mAbs. MFIs for RANTES and CD44 expression are depicted. (D) HeLa-CD4 cells, 3 days after transfection with indicated dsRNAs, were treated (+) for 30 minutes with 640 nM RANTES or were mock treated (–). Cell extracts were prepared, separated on 12% SDS-PAGE, and analyzed by immunoblotting for the activation of p44/p42 MAPK using anti-P-specific p44/p42 MAPK antibodies. The blots were then stripped and reprobed with anti-p44/p42 MAPK antibodies to confirm equal loading of samples.