Figure 6.

X-MAID thymocytes are unable to properly chemotax despite intact signaling. WT or X-MAID CD4 SP thymocytes were (A) placed in the upper chamber of a 5 μm pore transwell with 100ng/ml CCL19 in the media of the bottom chamber and incubated at 37 degrees for 2 hours. Cells in the bottom chamber were counted and percent migrated calculated. Graph shows results from 4 independent experiments, with each data point representing an average of three technical replicates from one experiment. Each experiment used pooled cells from 3-5 X-MAID mice, and cells from one matched WT littermate control. Data represent means ± StDev. Statistics were calculated using a Student’s t test, *p < 0.05. (B) WT or X-MAID CD4 SP thymocytes were left unstimulated, or stimulated with 100ng/ml CCL19 for the indicated times, permeabilized and stained with phalloidin to assess polymerized F-actin by flow cytometry. (C) Thymocytes stimulated as in B were fixed, lysed, and immunoblotted for pAkt (Ser473), pERK (Thr202/204), and total Akt and GAPDH as loading controls (left). WT and X-MAID samples were handled and analyzed in parallel. Blots were quantified by densitometry, and relative values were calculated after normalization to total Akt (right). For (B, C), each experiment used pooled cells from 3-5 X-MAID mice, and cells from one matched littermate control. Results are representative of 3 independent experiments.