H3k14ac

A subcellular protein fractionation kit for cultured cells (Thermo Fisher Scientific, Waltham, MA, USA; 78840) was used for fractionation experiments according to the manufacturer’s instructions. A 10 cm dish format was applied, which corresponded to a packed cell volume of 20 µL per well. Localization of SAMD1 was determined using a homemade SAMD1 antibody recognizing the SAM domain [9 (link)]. As loading controls for the respective fractions, a homemade SP1 antibody [18 (link)], anti-tubulin (Merck, Kenilworth, NJ, USA; MAB3408), and anti-H2Aub (Cell Signaling Technology, Danvers, MA, USA; 8240) were applied. For the detection of histone marks, the chromatin-bound fraction was used. H3 (Abcam, Cambridge, UK; ab1791), H3K4me2 (Diagenode, Denville, NJ, USA; C15410035), H3K4me3 (Diagenode, Denville, NJ, USA; C15410003) and H3K14ac (Abcam, Cambridge, UK; ab52946) antibodies were applied. The signal was quantified using the ImageLab software (v5.2.1, Bio-Rad, Hercules, CA, USA) and normalized to the H3 signal.

Western blot analysis was performed using as previously described (24 (link)). Briefly, mesenteric arteries were harvested and homogenized in ice-cold RIPA buffer with protease inhibitors. Equivalent amounts of protein (20 μg) were loaded onto 3−8% Tris-Acetate or 4−12% Bis-Tris gels (Invitrogen, USA) for electrophoresis and blotting. Membranes were incubated with the following primary antibodies: p-eNOS (1:2000, BD Biosciences), eNOS (1:2000, BD Biosciences), Nox4 (1:1000, Santa Cruz), SIRT1 (1:500, Cell Signaling Technology), H3K9ac (1:1000, Abcam), H3K14ac (1:1000, Abcam), H3 (1:1000, Abcam) and β-actin (1:2000, Santa Cruz) overnight at 4°C. Membranes were washed before incubating with horseradish peroxidase–conjugated secondary antibodies at room temperature. Immunoreactivity was visualized using enhanced chemiluminescence (ECL) and the Bio-Rad ChemiDOC XRS⁺ imaging system. Band intensity was quantified using ImageJ software.

Adult mutant or RNAi-treated worms were collected. Equal volume of sample buffer (0.1 M Tris pH 6.8, 7.5 M urea, 2 % SDS, 100 mM β-ME, 0.05 % bromophenol blue) was added to worms. Lysates were prepared by heating worms to 65 °C for 10 min, sonicating for two 30-s bursts, heating to 65 °C for 5 min, heating to 95 °C for 5 min and then kept at 37 °C until loading onto SDS-PAGE gel. Proteins were transferred to nitrocellulose and blotted with the following antibodies: H4K16ac (Millipore 07-329) at 1:250, H3K9ac (Abcam ab4441) at 1:1000, H3K14ac (Abcam ab5946) at 1:500, H3K56ac (Millipore 07-677) at 1:4000, H4K5ac (Upstate 07-327) at 1:500, H4K8ac (Abcam ab5823) at 1:2000, β-tubulin (Novus NB600-936) at 1:1000. Anti-MYS-1 antibodies were raised in rabbit against the N-terminal 28 amino acids (TEPKKEIIEDENHGISKKIPTDPRQYEK) and were used at 1:500.