Figure 7

Slit2N attenuates HIV-1 envelope-induced signaling pathway that mediates actin cytoskeletal reorganization. (A) Lysates from untreated, full-length Slit2 and Slit2N (3nM)-treated MT4 cells were stimulated with X4-tropic HIV-1 gp120 (10nM) for 10 minutes. The lysates were pre-cleared with sepharose beads and incubated with 20 μg/ml p21-activated kinase (PAK)-1 agarose for 60 minutes at 4°C. Agarose beads were collected by centrifugation and analyzed for Rac1-binding activity by Western blotting with anti-human Rac1 antibody (upper panel). Lysates probed with total Rac1 (lower panel) represent loading controls. (B) MT4 cells, pretreated with Slit2N, were treated with HIV-1 gp120 for various time points and lysed. To demonstrate LIMK1 activation, the lysates were immunoblotted for phospho-LIMK1. The blots reprobed with total LIMK1 antibody represented loading controls. (C) The same lysates were also immunoblotted with phospho-cofilin. Probing for total cofilin represented the loading control. (D) Resting primary T-cells, pretreated with Slit2N were treated with HIV-1 gp120 for various time points and lysed. To demonstrate LIMK1 activation, the lysates were immunoblotted for phospho-LIMK1. The blots reprobed with total LIMK1 antibody represented loading controls. (E) The same lysates were also immunoblotted with phospho-cofilin. Probing for total cofilin represented the loading control. All of the above experiments were repeated three times, and a representative experiment is shown.