Lysates were prepared using NaOH lysis followed by TCA precipitation [66]. TCA pellets were resuspended in SUME buffer (1% SDS, 8 M Urea, 10 mM MOPS, pH 6.8, 10 mM EDTA) and total protein concentration was determined using the BCA protein assay (Thermo Scientific). 10 µg total protein per lane was run on 10% tris-glycine polyacrylamide gels (PAGEr gold, Lonza) and transferred to Immobilon P membrane (Millipore) using a semi-dry transfer apparatus. Western blot analysis was performed by standard methods and developed with Supersignal West Pico (Thermo Scientific). Antibodies: Goat α-Sir2 (yN-19; Santa Cruz Biotechnology), mouse α-Sir2 (sc-25753; Santa Cruz Biotechnology) mouse α-Vma2 (Invitrogen), mouse α-Pkc1 (Invitrogen), and HRP-conjugated secondary antibodies (Jackson Immunoresearch Laboratories). Quantitation of Western blots was performed by densitometry using ImageJ comparing exposures that fell within a linear range of detection.
