Normal GPCR signaling but defective chemokine-activated adhesion in LAD PBLs. (A) Effect of SDF-1 on normal and patient PBL rolling and arrest on TNF-activated HUVECs under physiological shear flow. Leukocytes isolated from a healthy donor (control) or patient (LAD) were perfused for 1 minute at 0.75 dyne/cm² over HUVEC monolayers, and the number of accumulated leukocytes that either continued to roll (▦) or came to full arrest (▪) was determined. Transient tethers comprised less than 10% of the total cell-capturing events and were not included in the analysis. Results are given as mean ± range of determinations in 2 fields of view. (B) FACS staining of CXCR4 on control and LAD PBLs with the 12G5 mAb. Background staining is indicated by the thin black lines. (C) SDF-1 stimulation (100 nM, 30 seconds) of ERK1/2 phosphorylation in control or LAD PBLs. Immunoblotting with antiphosphospecific ERK1/2 (top panel) and anti-ERK (middle panel) is depicted. Cell lysates were separated on reducing 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Control actin immunoblotting (bottom panel) is shown. (D) IP-10, IL-2, and phorbol ester stimulation of Akt (Pkb) and ERK1/2 in T lymphoblasts. Blasts were stimulated with either IP-10 (100 nM, 30 seconds), IL-2 (1000 U/mL, 5 minutes), or PMA (100 ng/mL, 2 minutes) before lysis. Immunoblotting with antiphosphospecific Akt or ERK1/2 (first and third rows, respectively) and anti-Akt or ERK (second and fourth rows, respectively) is depicted. In panels C-D, lysates of agonist-stimulated lymphocytes were separated on reducing 10% SDS-PAGE.