Samples were resolved on SDS-PAGE followed by staining with GelCode blue reagent (Fisher)

Samples were resolved on SDS-PAGE followed by staining with GelCode blue reagent (Fisher). sister-chromatid cohesion defects and the resulting mitotic arrest caused by Naa50 depletion, indicating that NatA and Naa50 play antagonistic roles in cohesion. Purified recombinant NatA and Naa50 do not affect each other’s NAT activity had initially revealed the role of the NAT separation anxiety protein (San; also called Naa50 or Toll-Like Receptor 7 Ligand II NatE) in sister-chromatid cohesion (35). Mutation of Naa50 disrupted sister-chromatid cohesion and caused premature sister-chromatid separation and mitotic arrest. A subsequent study showed that this function of Naa50 was conserved in humans, as depletion of Naa50 in HeLa cells by RNA interference (RNAi) caused phenotypes consistent with cohesion defects, including premature separation of sister chromatids and mitotic arrest (36). Deletion of Nat5, the closest homologue of Naa50 in acetylation assays by incubating recombinant GST-Naa50 WT or F27A with [14C]acetyl-CoA (Fig. 1= 2 independent experiments. = 2 independent experiments. and quantified. = 2 independent experiments. Depletion of Naa50 in human cells causes defects of sister-chromatid cohesion, premature sister-chromatid separation, and subsequent mitotic arrest dependent on the spindle checkpoint (36). We first confirmed that Naa50 depletion indeed caused mitotic arrest in both 293FT and HeLa cells (Fig. 1, and that had been synchronized Toll-Like Receptor 7 Ligand II at telophase. DAPI, GFP, and tubulin staining was pseudo-colored = 2 independent experiments with 40 cells counted for each group in each experiment. before and at different times after photobleaching. The bleached areas are marked by hybridization (FISH) analysis with a probe that recognized the q RNF23 arm of chromosome 21 (Fig. 3regions are magnified in the value was calculated with Student’s test. = 2 independent experiments. = 3 independent experiments. We next performed metaphase spreads on mitotic Naa50 RNAi cells (Fig. 3, and where the closest homolog of Naa50 is not required for cohesion (Fig. 5and and and (Fig. 5and as determined by FACS. Results are the mean range, = 2 independent experiments. = 2 independent experiments. = 2 independent experiments. Naa50 and NatA Do Not Regulate each Other in Vitro We next tested whether Naa50 blocked the activity of NatA and vice versa (Fig. 7, and and and using the MLGP or SAGE peptides as substrates. Results are the mean S.D. of the reactions in triplicate. = 2 independent experiments. (49). Briefly, cell pellet was resuspended in Buffer A (10 mm HEPES, pH 7.9, 10 mm KCl, 1.5 mm MgCl2, 0.34 m Sucrose, 10% Glycerol, 0.1% Triton X-100, 1 mm DTT, and 1 protease inhibitor mixture (Roche Applied Science)). After an 8-min incubation on ice, the nuclear fraction was pelleted by centrifugation at 1300 for 5 min at 4 C and resuspended in SDS sample buffer. The supernatant was collected as the cytoplasmic fraction. Plasmid and siRNA Transfection Cells were treated with the desired Toll-Like Receptor 7 Ligand II plasmids with the Effectene reagent (Qiagen) according to the manufacturer’s protocols. Myc-Sgo1 and GFP-Smc1 stable cell lines were made by transfecting HeLa Tet-On cells with pTRE2 vectors encoding RNAi-resistant human Myc-Sgo1 and GFP-Smc1 and selected with hygromycin (Invitrogen). The surviving clones were picked and screened for the doxycycline-induced expression of Myc-Sgo1 or GFP-Smc1 using immunoblotting. For RNAi experiments, HeLa Tet-On cells were transfected with siRNAs with Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s protocols. The siRNA oligonucleotides used in this study are: Naa50 siRNA (5-GCUACAAUGACAAGUUCUAdTdT-3), Wapl siRNA (5-CGGACUACCCUUAGCACAAdTdT-3), Scc1 siRNA (5-GGAAGAAGCATTTGCATTGdTdT-3), Esco1 siRNA (Dharmacon ON-TARGETplus set of 4), Esco2 siRNA (Dharmacon ON-TARGETplus set of 4), Naa10 siRNAs (siGENOME D-009606-02, D-009606-03, Thermo Scientific), Naa15 siRNAs (siGENOME D-012847-01, D-012847-03, Thermo Scientific), Naa20 Toll-Like Receptor 7 Ligand II siRNAs (siGENOME D-008944-03,.