Binding of gp120 to CXCR4 results in a CD4-independent phosphorylation of Pyk2. Cell surface expression of CXCR4 and CD4 on CD4⁻ Jurkat cells was analyzed by flow cytometry (A), after staining of the cells with the anti-CD4 OKT4a MoAb (a), the anti-CXCR4 12G5 MoAb (b), or an isotype-matched control MoAb (c). CD4⁻ Jurkat cells were incubated with either gp120 wt (10 μg/mL), gp120 ▵HX1 (10 μg/mL), SDF1 (100 nmol/L), or the anti-CD3 UCHT-1 MoAb (10 μg/mL) for the indicated time periods at 37°C (B). Immediately after incubation, cells were lysed and Pyk2 was immunoprecipitated with an anti-Pyk2 Ab. The tyrosine phosphorylation status of Pyk2 was analyzed by immunoblotting using an anti-phosphotyrosine (anti-pTyr) MoAb. The immunoblot was stripped and reblotted with an anti-Pyk2 Ab (anti-Pyk2) to ensure that equivalent levels of Pyk2 were immunoprecipitated in each lane.