Figure 4.

CXCL10 regulates the activation of canonical NF-κB signaling through CXCR3. (a) 4T1 cells were cultured with the indicated doses of mCXCL10 for 24 h. Total cell lysates were subjected to western blotting with the indicated antibodies. (b) 4T1 cells were transiently transfected with NF-κB reporter vector for 6 h. After incubation with PBS or mCXCL10 for 24 h, the luciferase assay was performed (**P<0.01). (c) 4T1 cells were cultured with the indicated doses of JN-2 or AMG 487 for 24 h. Total cell lysates were subjected to western blotting with the indicated antibodies. (d) 4T1 cells were transfected with NF-κB reporter vector for 6 h. After incubation with DMSO or JN-2 (10 μM) for 24 h, luciferase assay was performed (*P<0.05). (e) 4T1 cells were cultured with DMSO or JN-2 (10 μM) for 1 h in serum-free medium and treated with mCXCL10 (300 ng ml⁻¹) for the indicated times. After nuclear and cytosolic fractionation, lysates were subjected to western blotting with the indicated antibodies. (f, g) 4T1 cells were transiently transfected with pcDNA3-Flag-empty or pcDNA3-Flag-P65 for 6 h. Transduced cells were further incubated with DMSO or JN-2 (10 μM) for 24 h. After culturing, cell lysates and CXCL10 protein secretion were analyzed by western blotting with the indicated antibodies (f) and ELISA (g; *P<0.05). (h) 4T1 cells were transfected with pcDNA3-Flag-empty (Veh) or pcDNA3-Flag-IκBα (Flag-IκBα) for 6 h. After incubation with fresh media for 24 h, cell lysates and CXCL10 protein secretion were analyzed by western blotting with the indicated antibodies (left) and ELISA (right; *P<0.05). (i) 4T1 cells were co-transfected with NF-κB reporter vector with pcDNA3-Flag-empty (Veh) or pcDNA3-Flag-IκBα (Flag-IκBα) for 6 h. After incubation with fresh media for 24 h, the luciferase assay was performed (**P<0.01).