Typhoon 9400 variable mode imager

Organoids or crypt-enriched fractions from mouse ileum were concentrated
by centrifugation, resuspended in 30% acetic acid, and sonicated. After
incubation overnight at 4 °C with agitation, samples were diluted 3-fold
with water. Insoluble material was removed by centrifugation at 100,000
× g for 2 h at 4 °C, protein concentrations of the supernatants
were determined by Bio-Rad Protein Assay (Bio-Rad), and equivalent amounts of
each sample were lyophilized. Lyophilized samples were dissolved in 5%
acetic acid and separated by 17% AU-PAGE^{14 (link)}. Folded Crp23 was created from a
synthesized 80% pure linear peptide (CPC Scientific) by the same
procedure as previously reported for the α-defensin HD5^{40 (link)}. Proteins were visualized with
SYPRO Ruby (Life Technologies). Gels were imaged using a Typhoon 9400 variable
mode imager (GE Healthcare). For western blot, samples were separated by
12.5% AU-PAGE and semi-dry transferred to nitrocellulose membranes.
Membranes were immediately fixed in glutaraldahyde and blocked in 5%
milk, before overnight RT incubation in rabbit anti-HD5 antibody (kind gift from
Edith Porter^{12 (link)}) at a 1:1000
dilution. Membranes were incubated in goat-anti-rabbit Alexa Fluor 488
(Invitrogen) and imaged using a Typhoon 9400 variable mode imager (GE
Healthcare).

In vitro mono-ADP-ribosylationassay was performed according to reported procedures^{17 (link)}. Briefly, 2.5 µM of SIRT6 WT, S56Y, G60A, R65A or H133Y was incubated in 30 µL of reaction mixture (25 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM DTT, 50 µM 6-alkyne-NAD) at 30 °C for 30 min. For negative control, 50 µM of NAD⁺ was added to replace 6-alkyne-NAD. Then click chemistry was performed. BODIPY-N₃ (1 µL of 4.5 mM solution in DMF), Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (1.8 µL of 10 mM solution in DMF), CuSO4 (1.5 µL of 40 mM solution in H₂O) and Tris(2-carboxyethyl)phosphine (1.5 µL of 40 mM solution in H₂O) were added into the reaction mixture. The click chemistry reaction was allowed to proceed at room temperature for 30 min. Then SDS loading buffer was added and the mixture was heated at 95 °C for 5 min. The reaction mixture was resolved by 12% SDS-PAGE. Protein gel was incubated in destaining buffer (50% methanol, 40% water, 10% acetic acid) at room temperature for 2 h. BODIPY fluorescence signal was then recorded by Typhoon 9400 Variable Mode Imager (GE Healthcare Life Sciences) with PMT 550 V and normal sensitivity. After recording the fluorescence, the gel was stained by Coomassie blue buffer (0.2% Coomassie brilliant blue R-250 dye, 50% methanol, 40% water, 10% acetic acid) to quantify the protein loading.