Patterns of phosphorylation of known intermediates of CCR7 intracellular signaling are altered in CDk5-deficient T cells. Cell surface expression of CCR7 on peripheral blood CD4⁺ and CD8⁺ T cells was examined by flow cytometry after labeling cells with CCL19-Fc followed by anti-human immunoglobulin G (IgG)-phycoerythrin (PE) and either CD4-APC or CD8-APC (A). Next, purified T cells from Cdk5^+/+C (Cdk5 WT) or Cdk5^−/−C (Cdk5 KO) mice were incubated with 100 ng/mL CCL19 for 0 to 4 minutes and then lysed with Triton X-100 buffer containing phosphatase/protease inhibitors. Total cell lysate (4 μg) was separated on a 4% to 12% Bis-Tris gel and transferred to nitrocellulose. Cell lysates were examined by western blot using antibodies against pERK1/2 to measure levels of phosphorylated protein and ERK1/2 to measure levels of pERK1/2 and total ERK1/2 loaded (B1). The relative increase in expression of pERK/ERK in lysates from either WT or KO T cells was determined at each time point using time 0 for respective samples (eg, 100%) as baseline (B2). The percentage reduction of pERK expression in KO T cells was also determined at each time point (B3). In separate experiments, purified T cells from WT or KO mice were again incubated with 100 ng/mL CCL19 and cell lysates were examined for expression of pMEK1/2 and GAPDH (C). Data shown are from 1 of 3 replicate experiments.