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. 2009 Jul 20;86(4):833–845. doi: 10.1189/jlb.0908551

Figure 5.

Figure 5.

MIP-1β induces Lyn, PI3K, and Pyk2 association and colocalization to form a multi-kinase signaling complex. Macrophages were treated without or with MIP-1β for 5 min prior to cell lysis. Cell lysates were immunoprecipitated with antibody specific for (A) Lyn, (B) PI3K, (C) Pyk2, or with control IgG. Immune complexes were resolved on SDS-PAGE and subjected to immunoblot with antibodies specific for Pyk2, PI3K, and Lyn. WCL of unstimulated cells served as a positive control for immunoblotting. (D) Macrophages were pretreated without or with the CCR5 antagonist M657 (1 μM) for 1 h prior to stimulation with MIP-1β for 5 min. Macrophages were fixed, permeabilized, and triple-labeled with antibodies to Pyk2, PI3K, and Lyn before examination by confocal microscopy. Subcellular distribution of Lyn (green; a, e, i, m), PI3K (blue; b, f, j, n), and Pyk2 (red; c, g, k, o) is shown in the single-channel images. Colocalization of Lyn, PI3K, and Pyk2 is indicated by arrowheads shown in the Merge images (white; d, h, l, p). Original scale bar = 10 μm (shown in d; applies to a–p).