Reduction of CXCR3 cell-surface expression following CXCL11 and PMA stimulation. (A) Time course of the reduction of CXCR3 expression in response to CXCL11 and PMA in 293-CXCR3 cells. CXCR3-expressing HEK 293 cells were trypsinized and incubated with either 1 mg/mL CXCL11 or 100 ng/mL PMA for the indicated time points at 37°C. The cells were then washed, immunostained with anti-CXCR3–specific antibodies, and FACS analyzed. Each value represents the mean plus or minus SE of 3 to 4 independent experiments. (B) Effect of PKC inhibitors on ligand- or PMA-induced CXCR3 down-modulation on the surface of 293 cells. 293-CXCR3 cells were trypsinized and incubated in medium alone or with a PKC inhibitor (staurosporin [0.5 μM], GF [5 μM], or rottlerin [5 μM]) for 30 minutes at 37°C, and then challenged with 1μg/mL CXCL11 or 100 ng/mL PMA for 1 hour at 37°C. The cells were then washed, and surface expression of CXCR3 receptors was analyzed. Each value represents the mean ± SE of 4 independent experiments. *P < .05 by ANOVA and Dunnett test for internalization induced by PMA with staurosporin versus without staurosporin, and with GF versus without GF. (C) The CXCR3-expressing HEK 293 cells, WT CXCR3, 349stopΔ20 C-tail truncated, 332stopΔ37 C-tail truncated, and 332-334L→A C-tail mutations were trypsinized and incubated with CXCL11 (1 μg/mL) or PMA (100 ng/mL) for 1 hour at 37°C. The cells were then washed, immunostained with anti-CXCR3–specific antibodies, and FACS analyzed. Each value represents the mean ± SE of at least 3 independent experiments. *P < 05 by ANOVA and Dunnett test for D37 CXCL11- and PMA-induced internalization versus WT.