Figure 5.

Toll-like receptor 7 signaling and IAV reduce the level of CYLD in alveolar macrophages. (A) MH-S cells were pretreated with or without z-VRPR-fmk (zVRPR, 100 µM) for 6 h before stimulation with or without imiquimod (Imiq, 1 µg/ml) for another 18 h. Cell lysates were analyzed by Western blotting with antibodies against CYLD, RelB, and regnase-1. Bar graphs show the levels of (B) CYLD, (C) RelB, and (D) Regnase-1 protein normalized against β-actin (n = 4, four independent experiments). Dark arrowhead points to total form of RelB or regnase-1, gray arrowhead to shifted band of regnase-1, empty arrowhead to cleavage form of RelB. (E) MH-S cells were pretreated with or without z-VRPR-fmk (zVRPR, 100 µM) for 6 h before stimulation with or without WSN virus (MOI = 0.25) for another 18 h. Cell lysates were analyzed by Western blotting with antibodies against CYLD and RelB. Bar graphs in (F) show the levels of CYLD and RelB protein normalized against β-actin (n = 3, three independent experiments). One representative experiment is shown in (A,E). (B–D,F) Relative intensity was calculated by dividing the intensity of indicated protein by β-actin and normalized against that of control (Ctrl). Data presented are the mean + SD; n.s., not significant; *p < 0.05, **p < 0.01, and ***p < 0.001 compared to control (one-way ANOVA followed by Tukey’s post hoc).