Anti sirt6

Cultured wild-type and Sirt6 cKO TECs were used to detect SIRT6 expression. TECs (CD45^–EPCAM⁺) isolated from wild-type and Sirt6 cKO mice were used to detect the expression of SPIB. After being washed with cold PBS, TEC were lysed in RIPA buffer (140 mM NaCl, 10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate and 0.1% SDS) complemented with a proteinase inhibitor cocktail (Sigma-Aldrich, P8340). Protein concentration was detected with Bradford assay. Proteins were analyzed by 10% SDS-PAGE and transferred onto PVDF membranes (Merck Millipore, IPFL00010). Each PVDF membrane was blocked with 5% non-fat dried milk (OXOID, LP0031) for 60–90 min at room temperature and incubated with each primary antibodies overnight on the shaking table at 4°C. After cleaning PVDF membrane with TBST solution for four times, the corresponding secondary antibodies were added for 45–60 min at room temperature. Protein bands were detected by chemiluminescence (Merck Millipore, WBKLS0500). ACTIN is used as internal reference for protein standardization. The primary antibodies used for western blot are as follows: Anti-SIRT6 (Cell Signaling Technology, 12486) diluted by 1:1,000; anti-SPIB (Cell Signaling Technology, 14337S) diluted by 1:1,000; anti-ACTIN (Sigma-Aldrich, A5441) diluted by 1:20,000.

Anti-NFATc4 (diluted 1:200, sc-13036), anti-phospho-NFATc4 (diluted 1:100, sc-32630) polyclonal antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, United States). Anti-SIRT6 (diluted 1:1000, 12486) antibody was bought from Cell Signaling Technology (Beverly, MA, United States). Anti-CaN A (diluted 1:1000, ab90540) antibody was purchased from Abcam (Cambridge, MA, United States). Anti-p38 (diluted 1:1000, 14064-1-AP) antibody was bought from Proteintech (Rosemont, IL, United States). Anti-α-tubulin (diluted 1:1000, T9026) and anti-Lamin B1 (diluted 1:1000, SAB1306342) antibodies were obtained from Sigma (St. Louis, MO, United States). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA, United States). PE was obtained from Tocris Bioscience (Bristol, United Kingdom). Rhodamine phalloidin and 4′6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen (Carlsbad, CA, United States). Other reagents were from Sigma-Aldrich unless otherwise stated.

Whole cell protein of untreated and 24 h Rapha Myr^® extract (0.5–1.25–2.5% v/v)-treated cells were prepared according to Laemmli (1970) and submitted to Western blot analysis, performed according to Grabowska et al., 2016 [72 (link)]. The primary antibodies used were: anti-integrin α5 (1:1000) (Immunological Sciences, Rome, Italy), anti-GAPDH (1:50,000) (Millipore, Darmstadt, Germany), anti-Poly (ADP-ribose)polymerase (PARP, 1:1000) (BD Biosciences, San Jose, CA, USA), anti-γH2AX Ser139 (1:1000) (Abcam, Cambridge, UK), anti-p53 (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p21 (1:500) (Sigma-Aldrich, St. Louis, MO, USA), anti-phospho-p53 Ser15 (1:250), anti-sirt 1 (1:250), anti-phospho-sirt 1 Ser47 (1:250), anti-sirt 3 (1:500), anti-sirt 5 (1:500), anti-sirt 6 (1:1000) and anti-sirt 7 (1:250) (Cell Signalling Technology, Denvers, CO, USA). Each protein target was detected by using specific secondary horseradish peroxidase-conjugated antibodies (1:2000) (Dako, Glostrup, Denmark) and an ECL system (Thermo Scientific, Rockford, IL, USA). The expression level of proteins was measured by densitometric analysis using the software Image J and GAPDH was chosen as the reference protein.

Using a cytoplasmic and nuclear protein isolation kit (Beyotime, P0028), nuclear extracts were obtained from chondrocytes. Chondrocytes were then lysed with non-denaturing NP-40 lysis buffer supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF), protease inhibitor cocktail, and phosphatase inhibitor cocktail (Sigma). An amount of 500 μg protein was immunoprecipitated with anti-Sirt6 (Cell Signaling Technology) or anti-total-STAT5 antibody (Cell Signaling Technology) and Protein A/G PLUS-Agarose (sc-2003, Santa Cruz Biotechnology) overnight at 4 °C. Beads with the immune-precipitated complex were washed with lysis buffer three times before denaturing in lysis buffer at 97 °C for 10 min. Nuclear extracts and immunoprecipitated protein were resolved by SDS-PAGE. Western blot was then performed.

Total protein was isolated from cells using cold radio immunoprecipitation assay buffer containing a protease inhibitor cocktail (Roche Applied Science, Penzberg, Bavaria, Germany). A total of 50 μg of protein was subjected to SDS-PAGE, and the blots were transferred onto a polyvinylidene difluoride membrane. After blocking, the membranes were incubated with anti-SIRT1 (Millipore, Temecula, CA, USA), anti-SIRT2 (Cell Signaling Technology, Danvers, MA, USA), anti-SIRT3 (Cell Signaling Technology), anti-SIRT5 (Millipore), anti-SIRT6 (Cell Signaling Technology), anti-eNOS (Cell Signaling Technology), anti-KLF2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-FOXM1 (Cell Signaling Technology), anti-β-actin (Santa Cruz Biotechnology), and anti-FLAG (Sigma-Aldrich) antibodies. Antibodies against cell cycle regulators and checkpoint molecules, including cyclin D1, cyclin D3, p18 INK4C, p21 Waf1/Cip1, p27 Kip1, CDK2, CDK6, phospho-RB, and phospho-p53 (Ser15), were purchased from Cell Signaling Technology. Immune-reactive protein bands were visualized by chemiluminescence using ECL reagents (GE Healthcare, Fairfield, CT, USA). Protein expression was imaged in a ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA, USA).