Figure 7.
Arrestins are involved in MIP-1β-stimulated macrophage chemotaxis and signaling complex formation. (A) WCL (10 μg) from unstimulated macrophages was subjected to immunoblot with an anti-arrestin antibody that recognizes Arr2 and Arr3. (B–F) Macrophages were treated without or with MIP-1β for 5 min prior to cell lysis. WCLs were immunoprecipitated with antibody specific for (B) Arr2/3, (C) ERK, (D) Lyn, (E) PI3K, (F) Pyk2, or control IgG. Immune complexes were resolved on SDS-PAGE and subjected to immunoblot with antibodies specific for Pyk2, PI3K, Lyn, Arr2/3, and ERK. (G) Macrophages were transfected with nontargeting control or Arr2/3 siRNA and then stimulated without (open bars) or with (solid bars) MIP-1β. Inset, Immunoblots were performed in parallel on the same batch of siRNA-transfected macrophages to determine Pyk2, PI3K, Lyn, Arr2/3, and β-actin protein levels. (H) Macrophages transfected with nontargeting control or Arr2/3 siRNA were stimulated without or with MIP-1β for 5 min prior to cell lysis. WCLs were immunoprecipitated with Lyn antibody or control IgG. Immune complexes were resolved on SDS-PAGE and immunoblotted with antibodies specific for Pyk2, PI3K, Lyn, Arr2, and ERK. Data shown are means ± se of three independent experiments with cells from different donors, along with representative blots (***, P<0.001).