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. Author manuscript; available in PMC: 2010 Aug 17.
Published in final edited form as: J Immunol. 2008 Nov 15;181(10):6730–6737. doi: 10.4049/jimmunol.181.10.6730

Figure 3. LPS induces Nrf2 activation in THP-1 cells.

Figure 3

A, THP-1 cells were stimulated with LPS (10 μg/ml) for various times and nuclear extracts prepared. Nuclear proteins were separated by SDS-PAGE and western blot analysis was conducted for Nrf2 protein expression. Chemiluminescent images were quantified by densitometry. The numerical values represent fold increase in expression following stimulation with LPS compared to no LPS, and normalized to β-actin as a loading control. B, Primary monocytes were stimulated with LPS for various times and cytosolic and nuclear extracts prepared. Extracts were separated by SDS-PAGE and Western blot analysis was conducted for Nrf2 protein levels. Cytosolic blots were reprobed with GAPDH and nuclear blots reprobed with Tata Binding Protein (TBP) to confirm sample loading. C, EMSAs were performed using LPS-stimulated nuclear THP-1 extracts and a biotinylated NQO1-ARE probe. D, Nuclear extracts from THP-1 cells stimulated for 2 h with LPS then preincubated with 1 μg of anti-Nrf2 or anti-p65 supershift antibodies, before EMSA. An unbiotinylated NQO1-ARE oligonucleotide probe was used as a cold competitor (as indicated).