Figure 4.

Chemotaxis of macrophages triggered by MIP-1β requires proline-rich tyrosine kinase Pyk2. (A) Macrophages were pretreated for 15–60 min with the upstream Pyk2 inhibitors dantrolene (10 μM) and AG17 (20 μM), inactive analog AG43 (20 μM), or control vehicle alone (0.1% DMSO) prior to stimulation with MIP-1β. (B) Macrophages were transfected with nontargeting control or Pyk2-specific siRNA followed by stimulation without (open bars) or with (solid bars) MIP-1β to induce chemotaxis. Immunoblots were performed in parallel on the same batch of transfected macrophages. (C) Macrophages were stimulated with MIP-1β for indicated times prior to cell lysis. Cell lysates were immunoprecipitated with anti-Pyk2 antibody or control IgG and immunoblotted with anti-phosphotyrosine (p-Tyr) antibody (upper panel) to assess Pyk2 activation. The blots were stripped and reprobed with anti-Pyk2 antibody to determine total Pyk2 protein (lower panel). (D) Macrophages were pretreated with the CaMKII inhibitor KN62 (1 μM), GSK3 inhibitor BIO (10 nM), or control vehicle alone (0.1% DMSO) for 1 h prior to stimulation with MIP-1β. Data shown are the means ± se of three independent experiments with cells from different donors, along with representative blots (***, P<0.001).