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. 2020 Oct 7;11:579266. doi: 10.3389/fimmu.2020.579266

Figure 9.

Figure 9

Canonical NF-κB signaling non-transcriptionally and non-translationally regulates IL-1β-induced ERK1/2 activation. (A) Cells were pretreated with the transcription inhibitor actinomycin D (Act D; 1 μM) and translation inhibitor cycloheximide (CHX; 100 μM) for 6 h and stimulated with IL-1β for 15 min. ActD and CHX had no effect on IL-1β-induced phosphorylation of ERK1/2, p65 and p105. Representative blots and relative levels of p- and t-ERK1/2, p65 and p105 have been illustrated in the left and right panels, respectively. (B) Cells were pretreated with the transcription inhibitor actinomycin D (Act D; 1 μM) and translation inhibitor cycloheximide (CHX; 100 μM) for 6 h and stimulated with IL-1β for 15 min. Subsequently, immunocytochemistry and co-localization analysis with nuclear staining dye TO-PRO-3 were performed. The nuclear localization of t-p65 was detected in the cells treated with IL-1β, whereas t-p65 showed cytoplasmic localization in control cells. IL-1β induced the nuclear localization of t-p65 in the presence of actinomycin D and cycloheximide. (C) Western blotting for the levels of p-ERK1/2, t-ERK1/2, t-p65 and t-p105 in cells transfected p65, p105, or scramble siRNAs. Decreased IL-1β-induced phosphorylation of ERK1/2 was observed in p65 or p105 siRNA-transfected cells. Representative blots for the levels of p-ERK1/2, t-ERK1/2, t-p65, and t-p105 and relative expression of p-ERK1/2 have been compared with the levels in cells transfected with scramble siRNA in the left and right panels, respectively. Results have been represented as mean ± SE from biological triplicates. *P < 0.05. Cell lysates (10 μg protein) were used for immunoblotting. β-actin was used as an internal standard (C). (D) Western blotting for the levels of p-MEK, p-ERK1/2 and p-p65 in the fraction precipitated with p-p65 and p-ERK1/2. Results have been represented as mean ± SE from biological triplicates. *P < 0.05. Cell lysates (50 μg protein) were used for co-immunoprecipitation experiments. IgG-heavy chain (IgG-HC) was used as an internal standard (D).