Fig 1: Mutation of the PD-L1 Cytoplasmic Domain Impairs G Protein Activation, ERK Phosphorylation, and F-Actin Polymerization in Response to CCL21(A) Diagram showing the signaling pathway of chemokine receptors, including CCR7 and CXCR4. Colored text indicates steps of the pathway interrogated, with red showing steps disrupted by loss of PD-L1 signaling and blue showing steps of the pathway without significant changes.(B) Quantification of IP-1 concentration in WT and Pdl1CyMt BMDCs following 30 min of CCL21 (1 µg/mL) treatment.(C) Western blot image using antibodies against ERK1/2, pERK1/2, p38, p-p38, and GAPDH at 0, 1, or 5 min following CCL21 (1 µg/mL) addition to BMDC media. The experiment was repeated twice with similar results.(D) Quantification of (C), showing fold change over no chemokine.(E) Representative flow plots of F-actin following 15 s of CCL21 (1 µg/mL) treatment in BMDCs.(F) Quantification of (E) showing fold change over no chemokine at 15s, 60 s, and 5 min after CCL21. n = 3 technical replicates per group. The data shown are combined from three independent experiments.(G) Representative flow plots of F-actin following 15 s of S1P (500 nM) treatment in BMDCs.(H) Quantification of (G) showing fold change over no chemokine. n = 3 technical replicates per group. The data shown are combined from two independent experiments.Statistical analysis was done using a one-way ANOVA (B and F) or an unpaired Student’s t test (D and H). *p < 0.05; **p < 0.01; ****p < 0.0001; n.s., p > 0.05. Error bars indicate standard error of the mean. See also Figure S6.