Enhanced and sustained CXCR4¹⁰¹³-mediated ERK1/2 activation depends on the SHSK motif. (A) A representative experiment out of 2 showing CXCL12-induced ERK1/2 activation in leukocytes from a healthy subject (CTRL) or a patient with WHIM harboring CXCR4¹⁰¹³, with (+) or without (−) 30 nM CXCL12 for 2 minutes at 37°C. Cell lysates were immunoblotted for ERK2 and phosphorylated ERK1/2, and data were quantified as P-ERK/ERK2 ratios. (B,C) Time course of CXCL12-induced ERK1/2 phosphorylation in transduced A0.01 T cells expressing similar amounts of CXCR4^wt, CXCR4¹⁰¹³, CXCR4^wt/Δi3, or CXCR4^1013/Δi3 (Figure 2A). Cells were stimulated with 30 nM CXCL12 for 2, 5, or 15 minutes, or unstimulated (0), and the ERK2 and phosphorylated ERK1/2 content in the cell lysates was analyzed as in panel A. Immunoblots for ERK2 and phosphorylated ERK1/2 from a representative experiment are depicted in panel B. The values in panel C (means ± SEM; n = 3) represent ERK1/2 activation quantified as P-ERK/ERK2 ratios in cells expressing CXCR4^wt, CXCR4¹⁰¹³, CXCR4^wt/Δi3, or CXCR4^1013/Δi3, expressed as a percentage of the ERK1/2 activation in CXCR4^wt-expressing cells at 2 minutes of stimulation (100%).