Figure 5.

Unc5CL-mediated NF-κB activation is IRAK-dependent but MyD88-independent. (a) HEK293T cells were transfected with the indicated siRNAs. Forty eight hours later, cells were transfected with NF-κB luciferase reporter gene plasmids together with an empty vector (EV) and the indicated expression constructs and were analyzed for NF-κB-dependent luciferase activity 24 h later. TLR2-transfected cells were additionally treated with 200 ng/ml Pam3CSK4. (b) Selected protein samples from experiments shown in (a) were probed by western blot using the indicated antibodies. (c and d) HEK293T cells were transfected with the indicated siRNAs. 48 h later, cells were transfected with the indicated expression constructs and supernatants were analyzed for IL-8 and CXCL1 secretion by ELISA. TLR2-transfected cells were additionally treated with 200 ng/ml Pam3CSK4. (a, c and d) Data represent the mean values±S.D. of technical triplicates; results are representative of three independent experiments. (e) HEK293T cells were transfected with the indicated expression constructs. Immunoprecipitates and extracts were analyzed by western blot using the indicated antibodies