1 siRNA pool (Dharmacon) utilizing RNAiMAX (Invitrogen) in line with guidelines in the supplier. Western Blotting–Cells had been washed with cold PBS (pH 7.4) after which scraped into lysis buffer containing 50 mM Tris-HCl (pH six.eight), 1 SDS, 1 mM PMSF, and protease inhibitor mixture. Lysates had been heated at 100 for 10 min and clarified by centrifugation. Equal amounts of protein have been subjected to SDSPAGE and immunoblotted with specific main antibodies. Anti-NMNAT1 mouse monoclonal antibody was bought from Santa Cruz Biotechnology. Rabbit polyclonal serum forJULY 19, 2013 VOLUME 288 NUMBERNMNAT1 Regulates rRNA TranscriptionFIGURE 1. NML interacts with NMNAT1. a, H1299 cells transiently transfected with FLAG-NML had been immunoprecipitated applying M2 beads. NML complexes were eluted with FLAG peptide, plus the co-precipitated proteins were identified by Coomassie Blue staining and mass spectrometry analysis of person bands. b, glutathione-agarose beads loaded with GST or GST-NML had been incubated with identical amounts of His6-NMNAT1, along with the captured NMNAT1 was detected by Western blotting (WB, appropriate panel). c and d, H1299 cells have been transfected with FLAG-NML and Myc-NMNAT1. Complex formation was analyzed by IP-Western blotting. e, U2OS cells stably infected with tetracycline-inducible NMNAT1 lentivirus had been analyzed by Myc IP and NML Western blotting. Third lane within the best panel shows the binding of physiological amount of Myc-NMNAT1 to endogenous NML.FIGURE two. NML promotes interaction amongst SirT1 and NMNAT1. a, H1299 cells had been transfected with FLAG-NML and Myc-NMNAT1 and subjected to glucose starvation for 18 h. The binding between Myc-NMNAT1 and endogenous SirT1 was detected by IP-Western blotting.Tacrine WCE, entire cell extract.SP187 b, H1299 cells have been transfected with all the indicated plasmids for 24 h and subjected to glucose starvation for 18 h.PMID:24118276 ChIP assay was performed to establish Myc-NMNAT1 binding to the rDNA promoter utilizing Myc antibody. c, H1299 cells have been subjected to glucose starvation for 18 h. ChIP assay was performed to identify endogenous NMNAT1 and SirT1 binding to the rDNA promoter.rRNA Biosynthesis Assay–HeLa cells treated with control siRNA, NMNAT1, or NML siRNA have been labeled with five Ci of [3H]uridine (38 Ci/mmol; PerkinElmer Life Sciences) for 30 min and washed twice with PBS. RNA was extracted with anRNeasy mini kit. For quantification of rRNA synthesis level, an identical quantity of total RNA was analyzed by liquid scintillation counting to figure out the incorporation of [3H]uridine.VOLUME 288 Number 29 JULY 19,20910 JOURNAL OF BIOLOGICAL CHEMISTRYNMNAT1 Regulates rRNA TranscriptionFIGURE 3. NMNAT1 regulates SirT1 activity and rRNA synthesis. a, p53-null H1299 cells had been transfected with all the indicated plasmids. Cells were treated with trichostatin A (150 ng/ml) for 5 h prior to harvest. The acetylation degree of p53 on K382 was determined by Western blotting. b, U2OS cells expressing endogenous p53 had been treated with handle or NMNAT1 siRNA for 24 h. Cells have been incubated with doxorubicin (0.5 M) for 16 h and with TSA (150 ng/ml) for 5 h just before harvest. Endogenous p53 Lys-382 acetylation level was detected by Western blotting. c and d, HeLa cells have been treated with NMNAT1 siRNA or NML siRNA and subjected to glucose starvation for 18 h. The price of rRNA synthesis was measured by [3H]uridine labeling. e, HeLa cells have been treated with NMNAT1 siRNA for 24 h followed by glucose starvation for 16 h. Pre-rRNA level was determined by RT-PCR and n.
