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. 2018 May 7;9:762. doi: 10.3389/fimmu.2018.00762

Figure 6.

Figure 6

SIRT1 activation was negatively correlated with intracellular domain of Notch (NICD) levels. (A) RAW264.7 cells transfected with SIRT1 siRNA or mock siRNA were treated with lipopolysaccharide (LPS) or PBS, and then RT-PCR and Western blotting were used to assess Hes1 mRNA and NICD protein levels. (B) Wild-type (WT) and sirt1−/− macrophages were treated with LPS or PBS and then Hes1 mRNA and NICD protein levels were examined using RT-PCR and Western blotting. (C) RAW264.7 cells were treated with LPS or PBS (control), and then the NICD acetylation level was evaluated by performing immunoprecipitation (IP) with anti-acetylation antibody and Western blotting with anti-NICD antibody. NICD expression in the whole-cell lysates was confirmed through Western blotting. (D) RAW264.7 cells were divided into control group (PBS), siSIRT1 group (SIRT1 siRNA + LPS), siSIRT1 + MG132 group (SIRT1 siRNA + LPS + MG132), and MG132 group (LPS + MG132), and NICD acetylation levels in the four groups were determined through IP with anti-acetylation antibody and Western blotting with anti-NICD antibody. SIRT1 expression in the whole-cell lysates was confirmed through Western blotting; n = 4. (E) Macrophages were separated into four groups and treated as in (D) and then NICD acetylation was determined by performing IP with anti-NICD antibody and Western blotting with anti-acetylation antibody. SIRT1 expression in the whole-cell lysates was confirmed through Western blotting; n = 3. (F) RAW264.7 cells were transfected with a plasmid expressing H363Y mutant SIRT1 or WT SIRT1, and then NICD acetylation was assessed through IP with anti-acetylation antibody and Western blotting with anti-NICD antibody. NICD expression in the whole-cell lysates was confirmed through Western blotting; n = 6. (G) WT and sirt1−/− macrophages were stimulated with LPS and then NICD acetylation was examined by performing IP with anti-acetylation antibody and Western blotting with anti-NICD antibody. NICD expression in the whole-cell lysates was confirmed through Western blotting. **p < 0.01 compared with LPS + mock siRNA group; n = 3.