Catalog no 89900

Eyes of 2.5-year-old fish were dissected and subsequently snap frozen in liquid nitrogen. Eyes were homogenized in RIPA buffer (Catalog No.: 89900 Thermo Fisher Scientific) and centrifuged supernatant was transferred to Eppendorf tubes and used for the Western Blotting. Proteins were resolved by SDS–PAGE, transferred to nitrocellulose membrane (Amersham GEHealthcare), and incubated with antibodies C4 (MAB1501R Sigma-Aldrich) for actin and α-Tubulin (DM1A) (Mouse mAb #3873) for tubulin and Acetyl-α-Tubulin (Lys40) (6-11B-1) for acetylated tubulin. For detection an Amersham ECL Western Blotting Detection Kit was used as described in manual. To visualize blots a ChemiDoc MP Imaging System was used and signals were measured using ImageJ.

PCa cell lines were lysed using radioimmunoprecipitation assay buffer (Catalog No. 89900, Thermo Fisher Scientific, Waltham, MA). Protein concentrations were measured by BCA assay (Catalog No. 23224, Thermo Fisher Scientific). Samples were separated via 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) for 90 min and transferred to polyvinylidene fluoride blotting membranes (0.2 μm pore size; GE Healthcare, Chalfont St. Giles, UK) for 2 h. The membrane was blocked in 5% bovine serum albumin for 2 h. Primary antibodies against SIRT1 (Catalog No. 8469S, Cell Signaling Technology Technology, Beverly, MA), SIRT2 (Catalog No. 12672S, Cell Signaling Technology), SIRT3 (Catalog No. 5490S, Cell Signaling Technology), SIRT4 (Catalog No. ab231137, Abcam, Cambridge, UK), SIRT5 (Catalog No. 8782S, Cell Signaling Technology), SIRT6 (Catalog No. 12486S, Cell Signaling Technology), SIRT7 (Catalog No. 5360S, Cell Signaling Technology) and succinyl-lysine (PTM Biolabs, Chicago, IL) were used to capture SIRT1-SIRT7 proteins overnight at 4 °C. After incubation with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h, the membranes were washed in Tris-buffered saline-0.1% Tween 20 (TBS-T) three times for 15 min. ECL prime Western blotting detection reagent (RPN2232, Citiva) was used to visualize the blots.