Molecules and Cells

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Fig. 3.

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Fig. 3. (A) NIH3T3 cells were seeded in 35-mm dishes. After serum starvation for 6 h, the cells were treated with 25 ng/ml human or mouse TNF-α and IL-1β, or 100 μM of TNFR2-SKE for 15 min as described in the Materials and Methods. DHA (1 mM) and TNF-α inhibitor (5 μM) were used as positive controls. Cell lysates were subjected to SDS-PAGE and IκB degradation was analyzed using western blotting. (B) Cell extracts were fractionated into nuclear and cytoplasmic fractions. Each fraction was analyzed for the localization of NF-κB/p65 using western blotting. Purity of each fraction was proven by co-analyzing β-tubulin (cytoplasmic) and RNA polymerase II (nuclear). Band intensity was quantified using ImageJ software. (C) Cells were pretreated with 1 mM of DHA for 1 h or with TNFR2-SKE for 0.5 h dose-dependently, and then treated with 25 ng/ml mouse TNF-α for additional 1 h. (D) Cells were irradiated with UVB (312 nm wavelength, 15 mJ/cm2), followed by treatment with TNFR2-SKE or DEX (10 μM) for 4 h. (C and D) In situ PLA was performed to analyze the interaction between NF-κB/p65 and NF-κB/p50. PLA signals in the cell population (n = 5) were quantified using the NIS Elements analysis. The average number of rolling-circle products (RCPs) per cell ± SD is shown. ***p < 0.001.
Mol. Cells 2019;42:151~160
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