Sc 365344

Cells cultured on coverslips were fixed in 4% of paraformaldehyde (Sangon Biotech) for 20 min, immersed in 0.1% of triton X‐100 (Sangon Biotech) for 15 min, followed by blocking in 5% of BSA for 1 h at room temperature. The combination of mouse anti‐SIRT7 (#sc‐365344, Santa Cruz) and rabbit anti‐phospho‐p65 (or rabbit anti‐acetyl‐p65) antibodies were employed concurrently to observe the co‐location of SIRT7 and phospho‐p65 (or acetyl‐p65), while mouse anti‐phospho‐p65 (#sc‐136548, Santa Cruz) and rabbit anti‐acetyl‐p65 were used for co‐labelling of phospho‐p65 and acetyl‐p65, respectively. All primary antibodies were used at 1:2000 dilutions. These primary antibodies were detected using Alexa Fluor 546 goat anti‐rabbit IgG (H + L) (Invitrogen) and Alexa Fluor 488 goat anti‐mouse IgG (H + L) (Invitrogen, used at 1:2000 dilutions) secondary antibodies. Nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI, Sigma–Aldrich). Images of the cells were obtained using a Nikon A1R microscope (Nikon, Minato City, Japan) with a fluorescence light source and filters.

SIRT1 and SIRT7 protein expression were studied by IHC. Sections (3μm thick) from the FFPE samples, mounted on glass slides, were deparaffinised in xylene and hydrated through a graded alcohol series. Antigen retrieval was accomplished by microwaving the slides in EDTA buffer (20′ and 40′ for SIRT1 and SIRT7, respectively) and endogenous peroxidase activity was blocked with 0.6% hydrogen peroxide.
Protein detection was performed using the NovolinkTM Max Polymer Detection System (Leica Biosystems, Nussloch, Germany), according to manufacturer instructions. Slides were incubated overnight at 4°C with mouse monoclonal antibodies specific for SIRT1 (#ab32441, 1:750, Abcam, Cambrige, United Kingdom) and SIRT7 (SC-365344, 1:100, Santa Cruz Biotechnology, Dallas, USA). The slides were washed, developed with diaminobenzidine chromogen and counterstained with hematoxylin. Finally, after dehydration and diaphanization, slides were mounted in Entellan^® (Merck-Millipore, Germany). Colorectal mucosa and kidney parenchyma sections were used as positive controls for SIRT1 and SIRT7, respectively.
Semi-quantitative assessment of SIRT1 and SIRT7 nuclear protein expression was done using Allred score [32 (link), 33 (link)], by estimating the proportion and intensity of positive cells (range 0, 2 to 8). Scores of 3 or greater were defined as positive.

Protein was extracted from fresh-frozen tissue using Kinexus Lysis Buffer (Kinexus Inc., Vancouver, Canada) and subsequently quantified using a Pierce BCA assay (Thermo Scientific Inc., Bremen, Germany), according to the manufacturer's instructions. For Western blot, 30μg of total protein of each sample was loaded in a 10% sodium dodecyl sulfate polyacrylamide gel for electrophoresis (SDS-PAGE). Proteins were blotted onto 0.2μm PVDF membranes (Bio-Rad Laboratories Inc., USA) and incubated overnight at 4°C with primary antibodies for SIRT1 (ab32441, 1:1000, Abcam, Cambridge, UK) and SIRT7 (SC-365344, 1:500, Santa Cruz Biotechnology, Dallas, USA). To ascertain equal loading of protein, the membranes were also probed with a monoclonal mouse antibody against β-Actin (clone AC-15, 1:8000, Sigma-Aldrich, CO., St. Louis, MO). The ClarityTM Western ECL Substrate (Bio-Rad, Hercules, CA, USA) was used to develop the membranes which were then recorded with Amersham Hyperfilm (GE Healthcare Buckinghamshire, UK). Protein band optical densities were determined by scanning and analyzing ECL signals in the linear range using Bio-Rad Image Lab 5.2.1 software. Values were normalized to the level of β-Actin in each sample.