Rapid Gαi-independent phosphorylation of Jak2 after stimulation with CCL21 in primary mouse lymphocytes.

Freshly isolated or activated mouse lymphocytes were exposed to CCL21 for indicated times, and lysates were immunoprecipitated with anti–PTyr Ab and developed in Western blot with anti–Jak2 Ab (WB:JAK2). (A) CCL21-induced Jak2 phosphorylation in freshly isolated lymphocytes. The arrow indicates the position of Jak2. At the bottom of the blot, a nonspecific band is shown to indicate equal loading of protein in each lane. (B) CCL21-induced Jak2 phosphorylation in activated lymphocytes. Activation was carried out as described in “Materials and methods.” As a control for equal amount of Jak2 in each lysate, 15 μg of each lysate was directly developed with anti–Jak2 Ab. (C) CCL21-induced Jak2 phosphorylation can be blocked by Tyr AG490 but not by PTX. Activated lymphocytes were exposed to CCL21 for indicated times after pretreatment for 2 hours with either PTX (0.1 μg/mL) or Tyr AG490 (100 μM). Equal loading was controlled as described in the legend to panel B. MW indicates molecular weight (kDa); ip, immuno-precipitating Ab.