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This work was supported by Ministry of Education, Culture, Sports, Science and Technology (MEXT)/Japan Society for the Promotion of Science (JSPS) KAKENHI [JP17H01522/JP17K19479/JP20H00489 (M

Posted on March 15, 2022 by president2010

This work was supported by Ministry of Education, Culture, Sports, Science and Technology (MEXT)/Japan Society for the Promotion of Science (JSPS) KAKENHI [JP17H01522/JP17K19479/JP20H00489 (M.K.), JP19KK0179 (M.K., K.Y., and S.A.K.), JP18H04536 (K.Y.), JP18H05534 (H. lysines in eDHFR using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. Donor treatment only did not significantly increase acetylation ( 5% yield) in all the lysine residues in nucleosome and eDHFR (and and and were acquired using a high 5-(N,N-Hexamethylene)-amiloride excitation power condition (50% of 20-mW 488-nm and 5% of 20-mW 561-nm laser lines, each 100-ms exposure time). For Fig. 2= is the relative intensity; is the recovered fraction; is the binding coefficient; is the baseline (unbleached fraction)] (26). In-Cell Histone Acetylation. HeLa S3 cells were treated with 100 M BSO in growth medium at 37 C for 1 d; 0.5 mM PEG-LANA-DSSMe was preincubated with 10 mM NAC-Ac, 2 mM TCEP, and 0.5 mg/mL Dextran-tetramethylrhodamine in PBS for 10 min. The preincubation mixture was incorporated into the cells by bead loading (15), and the cells were incubated in growth medium containing 30 mM NAC-Ac and 100 M BSO at 37 C for indicated time. The cells harvested with accutase (Innovative Cell Technologies) were incubated with 1 g/mL DAPI in growth medium at r.t. for 15 min to stain dead cells, and sorted by FACS Arial II or III (BD Biosciences). The living cells (DAPI negative) were classified into three groups with their tetramethylrhodamine signals: low population have the same signal as nontreated cells, high population have the highest about 5% intensity of the sample, and remaining cells were classified as mid population. Histones were isolated with Histone Purification Kit (Active Motif) according to the manufacturers instructions. In brief, histones were extracted with acid, and the H2A/H2B and H3/H4 fractions were isolated by subsequent elution. For LC-MS/MS analysis, the histones were dissolved in 20 L MQ. To the histone solution, 20 L of 200 mM NH4HCO3 aq., 40 L of propionic anhydride solution was added, and pH was adjusted to 8 by adding ammonia solution. 5-(N,N-Hexamethylene)-amiloride After 30-min incubation at r.t., the solvents were removed by Speed-Vac evaporator. Then, the samples digested by Trypsin Gold and Glu-C as described in in and analyzed by LC-MS/MS. Sample Preparation for Western Blotting. Histones were isolated from the cells by Histone Purification Kit according to the manufacturers instructions or by acid extraction. The histones were dissolved in MQ and pH was adjusted by adding 1 M Tris?HCl (pH 7.5). To 5-(N,N-Hexamethylene)-amiloride equalize the concentrations of the histones, the samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by oriole staining (Bio-Rad), and the signals were recognized by BioDoc-It Imaging System and measured by Fiji/ImageJ Version 1.51h. After equalizing the histone concentration, the histones were analyzed by Western blotting. Antibodies. Rabbit polyclonal antibodies against H3 (abcam, ab1791), H3K9ac (Merck, 07C352), H3K18ac (abcam, ab1181), and H2B (abcam, ab1790), rabbit monoclonal antibodies against H2B ubiquitination (Cell signaling technology, 5546S), and a mouse monoclonal antibody against H2B (abcam, ab52484), and H2BK120ac were used for Western blotting. For generating monoclonal antibody against H2BK120ac, H2BK120ac-containing synthetic peptide (NH2-CSEGTKAVTK(Ac)YTSSK-CONH2) was coupled to keyhole limpet hemocyanin and used to immunize mice (27); after generating hybridomas, clones were screened by enzyme-linked immunosorbent assay (ELISA) using plates coated with the revised or unmodified peptide conjugated with BSA. After recloning, supernatants from clones reacting with the H2BK120ac-containing synthetic peptide were used to probe blots using recombinant nucleosomes, in which H2BK120 was acetylated by LANA-DSH and acetyl-CoA (11), and the ones giving a single band at Rabbit polyclonal to ABCG5 the size of histone H2B were selected. Hybridoma cells were routinely cultivated in GIT medium (Fujifilm, 637C25715), supplemented with 1 ng/mL IL-6 (FunaKoshi, 206-IL-010) and 100 g/mL Gentamicin (Sigma, G1397-10 mL), at 37 C in an atmosphere of 5% CO2. For antibody purification, 5-(N,N-Hexamethylene)-amiloride the supernatant of hybridoma cell tradition was applied to 1 mL.

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