Figure 5-6923.

The effects of overexpression of EYFP-p62WT or EYFP-p62ΔUBA on RANKL-induced NF-κB and NFAT-dependent transcription. RAW264.7 cells stably expressing EYFP-p62WT or EYFP-p62ΔUBA were transfected with the 3-kb Luc-SV40 NF-κB luciferase reporter plasmid (A) or pNFAT-TA Luc reporter gene (B) and treated with 100 ng/ml of RANKL for various time periods. After stimulation, cells were lysed and luciferase activities in lysates determined. Each bar is the mean ± SEM from triplicate wells. Representative results from three independent experiments are shown. *The P values of the effect of RANKL stimulation compared to its respective controls (***P < 0.001, **P < 0.01, *P < 0.05). ^#The P values of the effect of overexpression of pEYFP-62WT and pEYFP-62ΔUBA on RANKL-induced NF-κB activity compared to pcDNA3.1-transfected control (^###P < 0.001). C–D: Effect of EYFP-p62WT and EFYP-p62ΔUBA overexpression on RANKL-induced NFAT protein expression in RAW stable cell lines. RAW264.7 cells stably transfected with pcDNA3.1, EYFP-p62WT, and EFYP-p62ΔUBA were stimulated with 100 ng/ml of RANKL for the indicated times. After stimulation, whole cell extracts were analyzed for NFAT protein expression by Western blotting. The same membrane was stripped and reprobed with an antibody for β-tubulin, which served as an internal control for differences in loading and transfer. C: The levels of NFAT and β-tubulin proteins are shown. D: NFAT protein levels are shown as the ratio of NFAT to β-tubulin. Each bar is the mean ± SEM from representative results from three independent experiments. *The P values of the effect on RANKL-induced NFAT protein synthesis at different time points compared to its unstimulated control (***P < 0.001). ^#The P value compared to the pcDNA3.1 control (^##P < 0.01, ^###P < 0.001).