Fig 1: The ROS–mediated inhibition of Sirtuin-1, but not Sirtuin-2, may be responsible for inflammation.(A) A fluorescence-based (EX: Em = 355/460 nm) enzyme assay of sirtuin-1 (SIRT1) in splenic Mφ cells. The assay was performed as instructed by the manufacturer (BPS Biosciences) using a cell lysate containing 1 μg of total protein. (B) Fluorescence-based sirtuin-2 assay per the manufacturer’s protocol (Abcam) with 1 μg of protein derived from the cell lysate of Mφ cells. (C) IB analyses followed by (D) densitometric analyses of sirt-1,2, acetylated p65, and β-actin in splenic Mφ cells of NTg and Tg+/−ATG13 mice (n=3 pr group). Dual IF analyses of SIRT-1 (red) and 3-nitrotyrosine (3NT; green) in the spleen of 10–12 weeks ‘old (E) NTg and (F) Tg+/−ATG13 mice (n=6/group). (G) 3D visualization of a single cell (ImageJ) displays the juxtaposition of SIRT1 and 3NT. (H) Quantification followed by scatter boxplot analysis, analyzing the total number of 3NT-ir puncta per cell in 19 cells /group. Unpaired t test indicates ****p<0.0005 versus control. (I) Tg splenic macrophages were transfected with siRNA against Sirt1 (250 pmol; Cat # AM16708; ThermoFisher Scientific, MA), followed by treatment with 0.5 mg/mL LPS for 2 hrs, and then immunostained with sirt1 antibody to check the efficiency of siRNA. Enzyme activity assay of (J) SIRT1 and (K) SIRT2 after 30 minutes of incubation with siRNA- transfected and non-transfected cell lysates (1 μg protein).(L) IF analysis of acetylated p65 in Tg splenic Mφ cells transfected with sirt1 siRNA followed by the treatment with 0.5 mg/mL LPS. Results are the mean ± SEM of three different experiments.
Fig 2: The ROS-mediated inhibition of Sirtuin-1, but not Sirtuin-2, may be responsible for inflammation. A A fluorescence-based (excitation: emission = 355/460 nm) enzyme assay of sirtuin-1 (SIRT1) in splenic Mφs. The assay was performed as instructed by the manufacturer (BPS Biosciences) using a cell lysate containing 1 µg of total protein. B Fluorescence-based sirtuin-2 assay performed according to the manufacturer’s protocol (Abcam) with 1 µg of protein derived from the cell lysate of Mφ cells. C IB analyses followed by D densitometric analyses of Sirt-1/2, acetylated p65, and β-actin in splenic Mφ cells from NTg and Tg+/-ATG13 mice (n = 3 per group). Dual IF analyses of SIRT-1 (red) and 3-nitrotyrosine (3NT; green) in the spleens of 10- to 12-week-old E NTg and F Tg+/-ATG13 mice (n = 6/group). G 3D visualization of a single cell (ImageJ) showing the interaction between SIRT1 and 3NT. H Quantification followed by scatter boxplot analysis of the total number of 3NT-ir puncta per cell in 19 cells/group. The unpaired t test indicates ****p < 0.0005 versus the control. ITg splenic macrophages were transfected with siRNA against sirt1 (250 pmol; Cat # AM16708; Thermo Fisher Scientific, MA), treated with 0.5 mg/mL LPS for 2 h, and then immunostained with a Sirt1 antibody to determine the efficiency of the siRNA. Enzyme activity assay of J SIRT1 and K SIRT2 after 30 min of incubation with siRNA-transfected and non-transfected cell lysates (1 µg protein). L IF analysis of acetylated p65 in Tg splenic Mφ cells transfected with Sirt1 siRNA and then treated with 0.5 mg/mL LPS. The results are presented as the means ± SEMs of three different experiments
Supplier Page from Abcam for SIRT2 Activity Assay Kit (Fluorometric)