R5- or X4-tropic HIV-1 gp120 promotes p38 kinase phosphorylation in a CD4-independent manner.

Cell surface expression of CCR5 of CD4⁻ Jurkat cells was analyzed by flow cytometry (A), after staining of the cells with an isotype-matched control MAb (i) or the anti-CCR5 182 MAb (ii). CD4⁻/CXCR4⁺ Jurkat cells (B), CD4⁻/CCR5⁺ Jurkat cells (C), or CD4⁻/CCR5⁺ CEM cells (D) were incubated in medium alone or in the presence of gp120 JRFL (10 μg/mL), MIP-1β (100 nM), recombinant soluble CD4 (10 μg/mL), anisomycin (500 ng/mL), PMA (1 μM), gp120 HXB2 (10 μg/mL), SDF-1α (100 nM), gp120-CD4s complexes (molar ratio 1:20), or recombinant soluble CD4 (10 μg/mL) for the indicated time period at 37°C. Immediately after incubation, cells were lysed; proteins were separated by SDS-PAGE and analyzed by immunoblotting with anti-p38 kinase antiserum to verify equal loading and efficiency of protein transfer.