X4 gp120 and chemokines induce macrophage Pyk2 tyrosine phosphorylation.

Macrophages were stimulated for 5 minutes with 2.5 μg/mL X4 (IIIB) and R5 (JRFL) gp120 (A), or stimulated for the indicated time periods with 200 nM of the chemokines MIP-1β or SDF-1α (B). Cell lysates were resolved by 8% SDS-PAGE, transferred to a nitrocellulose membrane, and subjected to immunoblot analysis with an antibody specific for the phosphorylated form of Pyk2 (upper panels, P-Pyk2). The same blot was then reprobed with an antibody specific for total Pyk2 (lower panels). Each experiment was repeated at least 4 times and the presented blots are representative of these experiments. To test the effect of different agonist concentrations, macrophages were stimulated for 5 minutes with the indicated concentrations of gp120 (C) and MIP-1β (D), and analyzed by immunoblot as described above.