Anti sirt6 antibody

For histological analyses, mice were euthanized and the hearts were cut into transverse segments, fixed in 10% formaldehyde, and embedded in paraffin. Sections were cut at a thickness of 10 μm and either stained with Masson's trichrome to measure fibrotic areas using ImagePro software. The following antibodies were used for immunohistochemistry analyses: anti-Ly-6G (1:100, Abcam) to detect neutrophils and anti-CD68 (1:200, Abcam) to detect macrophages. Quantitative assessment of neutrophil and macrophage density was performed by counting the number of Ly6G- and CD68-immunoreactive cells, respectively, in a double-blind fashion from five different fields of the infarcted area at 400 × magnification. For immunofluorescent staining, tissue sections were incubated with anti-SIRT6 antibody (1:100, Abcam) overnight and secondary antibody for 90 min. DAPI solution was then added for 2 min to stain the nuclei. Images were captured using a Laser-Scanning Confocal Microscope (Olympus FluoView™ FV1000, Tokyo, Japan).

The cultured cells were treated with tissue lysate buffer (Beyotime, China). After centrifugation at 10,000×g and 4 °C for 20 min, the supernatant was collected, separated on 10–12% SDS-PAGE gels, and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Anti-Sirt6 antibody (Abcam, USA) was used to detect the corresponding target protein expression. After incubation with HRP-conjugated secondary antibody, the immune signals were detected with a Western chemiluminescent HRP substrate (Millipore).GAPDH was used as an internal reference to normalize the expression levels of the target proteins. A commercial antibody against GAPDH was obtained from Abcam.

NCI-H23 and A549 cells were lysed with RIPA lysis solution (Beyotime) containing a mixture of protease inhibitors. The cell protein lysis product was separated by 10% SDS-PAGE electrophoresis, transferred onto a 0.22 mm PVDF membrane (Millipore), and detected with a specific antibody. A chemiluminescent substrate was added to the specific strip and quantified with densitometry (Quantity One software, BioRad). The GAPDH antibody was used as a control. Anti-Caspase-3 antibody, cleavage Caspase-3, poly (ADP-ribose) polymerase, cleavage PARP, BAX, BAK, cyclin D3 and CDK4 (1:1000) were purchased from Cell Signaling Technology. Anti-SIRT6 antibody was purchased from Abcam. BCL-2 and BCL-XL antibody were purchased from Proteintech.