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. 2015 Nov;88(5):894–910. doi: 10.1124/mol.115.099960

Fig. 8.

Fig. 8.

Changing canonical to noncanonical amino acids diminishes CCR6 activity in HEK-293T and Jurkat E6-1 cells. (A) HEK-293T cells were transfected with variable amounts of DNAs encoding wild-type and mutant CCR6-YFP receptors to produce equal surface expression. Migration was measured using a microchemotaxis chamber containing CCL20 in the lower wells, as described in Materials and Methods. The data shown are the mean ± S.E.M. from four experiments. The asterisks indicate a significant difference from HEK-293T cells expressing the wild-type receptor and, as indicated by the bar, between cells expressing CCR6-D142Y and CCR6-D142Y/A146V. (B) Cells transfected as in (A) were loaded with Fura-2/AM and assayed for intracellular calcium mobilization on a spectrofluorometer in response to 1 μg/ml CCL20, as described in Materials and Methods. The data shown are the mean ± S.E.M. of three experiments. The asterisks indicate a significant difference from cells expressing the wild-type receptor and, as indicated by the bar, between cells expressing CCR6-D142Y and CCR6-D142Y/A146V. (C) Jurkat E6-1 cells were transfected with variable amounts of DNAs encoding wild-type and mutant CCR6-YFP receptors to produce equal surface expression. In each experiment, the means were obtained for the percentage of input cells migrating to duplicate lower wells of a Transwell plate, as described in Materials and Methods. The data shown are the mean ± S.E.M. from three experiments. The asterisks indicate a significant difference from Jurkat E6-1 cells expressing the wild-type receptor or between the cells connected by the horizontal bars. (D) Cells transfected as in (C) were loaded with Fura-2/AM and assayed for intracellular calcium mobilization on a spectrofluorometer in response to 1 μg/ml CCL20, as described in Materials and Methods. The data shown are the mean ± S.E.M. of three experiments. The asterisks indicate a significant difference from cells expressing the wild-type receptor or, as indicated by the bar, between cells expressing CCR6-D142Y and CCR6-D142Y/A146V. *P < 0.05; **P < 0.01; ***P < 0.001.