Fig. 5

SMYD5 promotes inflammatory response by activating NF-κB signaling pathway. A FLS were treated with IL-1β (10 ng/ml) for indicated times, and cell lysates were analyzed using phosphor-specific antibodies for p65, p-IKKα/β, and p-IKBα to assess NF-κB signaling activation. (B–D) FLS were pretransfected with siNC or siSMYD5 and then incubated with 10 ng/ml IL-1β for 15 min. B Immunoblot analysis of whole-cell lysates for expression and phosphorylation of key NF-κB subunits. Right panel: quantification of p65 phosphorylation (n = 6) and p-IKKα/β and p-IKBα expression (n = 8). C Immunoblot analysis of nuclear and cytoplasmic p65 distribution. p65 expression was normalized to Lamin A/C (nuclear) or GAPDH (cytoplasmic), n = 5. D Immunofluorescence staining to assess nuclear translocation of p65. Scale bars, 50 μm. E–G FLS pretransfected with or without LV-SMYD5 were treated with 10 ng/ml IL-1β for 15 min. E Immunoblot analysis of p65 phosphorylation. p-p65 expression was normalized to total p65, n = 3. F Immunofluorescence staining to examine p65 distribution between nuclear and cytoplasmic compartments. Scale bars, 100 μm. G Immunoblot analysis of nuclear and cytoplasmic p65 distribution. p65 expression was normalized to Lamin A/C (nuclear) or GAPDH (cytoplasmic), n = 5. Data presented as mean ± SEM, ***p < 0.001, at least three independent experiments were repeated. Data presented as mean ± SEM, p-Values were calculated by two-tailed Student’s t-test (G) or one-way ANOVA test (B, C, E), *p < 0.05, **p < 0.01, ***p < 0.001, n.s. means no significance