Sirt1 clone 3h10.2

Whole-cell extracts of proliferating or quiescent HUVECs were prepared by cell lysis in CHAPS buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 1% CHAPS) complete protease inhibitor cocktail on ice for 20 min. Proteins were separated by SDS–PAGE prior to transfer onto nitrocellulose membranes (Protran; Schleicher & Schuell, Dassel, Germany). The following primary antibodies were used in Western blotting: human caspase-9 and caspase-3 (clone 8G10; BD Biosciences), Sirt1 (clone 3H10.2; Millipore), PKM1 (Proteintech), PKM2 (clone D78A4; Cell Signaling), XIAP (clone 48; BD Biosciences), LC3B (clone D11; #3868; Cell Signaling).

The following antibodies were used: mouse monoclonal SIRT1 7B7 (Novus Biologicals, LLC, Littleton, CO, USA) for immunofluorescence staining and mouse monoclonal SIRT1 Clone 3H10.2 (Millipore, Merck KGaA, Darmstadt, Germany), rabbit polyclonal SIRT1 (M07-131; Millipore, Merck KGaA) for immunoblotting, RPS19BP1 (AROS, ATLAS Antibodies AB, Stockholm, Sweden), PARP and p53 phospho-Ser46 (BD Pharming, Pharmingen, San Diego, CA, USA), p53 DO-1, GFP FL and PML H-238 (Santa Cruz Biotechnologies Inc., Dallas, TX, USA), Flag M2 (Sigma-Aldrich, St Louis, MO, USA), actin C4 (MP Biomedicals, (LLC, Santa Ana, CA, USA)), HA clones 12CA5 and 3F10 (Roche, F. Hoffmann-La Roche Ltd., Basel, Switzerland), p53 phospho-Ser46, SIRT1 (rabbit Ab), SIRT1 phospho-Ser27 (all Cell Signaling Technologies Inc., Danvers, MA, USA), acetyl-Lys373/382 p53 (Millipore, Merck KGaA). Affinity-purified rabbit HIPK2 antibodies have been described previously.^{33 (link), 39 (link)} Phospho-specific SIRT1 pSer682 antibodies were generated by immunizing rabbits with the following KLH-coupled peptide: NH₂-SGTCQ(pS)PSLEC-CONH₂. Rabbit sera were affinity-purified against the phospho-peptide and subsequently non-phosphorylation specific antibodies were removed by a column containing the immobilized non-phospho-peptide. The rabbit SIRT1 phospho-Ser682 antibody will be commercially available soon from Millipore (Merck KGaA).