RANTES activation of p44/p42 MAPK. (A) Serum-starved primary CD4⁺ T cells were treated with 50 nM or 640 nM RANTES for 30 minutes and analyzed for p44/42 MAPK activation. WCEs were separated on 12% SDS-PAGE gels and immunoblotted using phospho-specific anti-p44/42 pAbs. The blots were then stripped and reprobed with anti-p44/p42 pAb to confirm equal loading of samples. (B) Serum-starved primary CD4⁺ T cells were pretreated with pertussis toxin (Ptx, 500 ng/mL) for 6 hours, then incubated with 640 nM RANTES for 30 minutes and analyzed for p44/42 MAPK activation as described. (C-D) Serum-starved HeLa-CD4 cells were treated for 30 minutes with RANTES (640 nM) or [⁴⁴AANA⁴⁷] RANTES (640 nM) or mock treated. (C) WCEs were separated on 12% SDS-PAGE gels and analyzed for p44/42 MAPK activation as described. (D) WCEs were separated on 12% SDS-PAGE gels and immunoblotted using anti-FAK pAbs. The blots were then stripped and reprobed with anti-FAK serum to confirm equal loading of samples. (E) Primary PHA-activated CD4⁺ T cells were incubated for 24 hours with 640 nM RANTES or mutants Met-RANTES, Met-RANTES-E66S, or [⁴⁴AANA⁴⁷] RANTES and activation of p44/p42 MAPK analyzed as described. One of 2 to 4 individual experiments is shown.