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Figure 3 | Journal of Neuroinflammation

Figure 3

From: Neuronal RING finger protein 11 (RNF11) regulates canonical NF-κB signaling

Figure 3

Prolonged nuclear p65 translocation after TNF-α stimulation observed in neuronal cells with knockdown of RNF11. (A) SH-SY5Y cell lines (untransduced, small hairpin RNA (shRNA)-RNF11, shRNA-Scramble) were stimulated with TNF-α for 0, 30, 60 or 120 minutes. Cells were immunostained for p65 (red) and nuclear DNA (Hoechst 333258, blue). Representative images obtained at 0, 30 and 120 minutes after stimulation are shown. (B) Cells were analyzed for overlap of p65-positive and Hoechst 333258-positive pixels. The fold change in percentage overlap of p65- and Hoechst 333258-positive pixels was calculated relative to unstimulated conditions. (C) Murine primary cortical neurons were treated with cytosine arabinoside and transduced with lentiviruses containing shRNA targeted against RNF11 (shRNA-RNF11 neurons) or scramble shRNA sequence (shRNA-Scramble neurons). Quantitative RT-PCR (qRT-PCR) was used to measure relative RNF11 mRNA levels. (D) shRNA-RNF11 or shRNA-Scramble neurons were stimulated for 0, 30 or 120 min before being immunostained for p65 and Hoechst 333258. Transduced cells were analyzed for overlap of p65- and Hoechst 333258-positive cells. The fold change in percentage overlap of p65- and Hoechst 333258-positive pixels was calculated relative to unstimulated conditions. (E) shRNA-Scramble and shRNA-RNF11 cells were stimulated for 0, 30 or 120 minutes. Cytoplasmic and nuclear fractions were resolved by SDS-PAGE. (F) ImageJ software was used to quantify the ratio of the densitometry of the p65 bands in the nuclear fractions to the density of histone 1 immunoreactivity. The ratio for each time point was compared relative to the steady-state ratio, which was set at 100%. (G) ImageJ software was used to quantify the ratio of the densitometry of the p65 bands in the cytoplasmic fractions in a manner similar to that described in (F). All p65 colocalization values are means ± SEM of triplicate experiments. qRT-PCR values are expressed as mean comparative cycle threshold ± SEM of triplicate experiments. Western blots of cytoplasmic and nuclear fractions were run from triplicate experiments. ** P < 0.01; *** P < 0.001.

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