Hole-cell extracts in 1?Laemmli buffer had been electrophoresed on an eight?six (wt/vol) Tris lycine gel (Life Technologies), electroblotted onto a nitrocellulose membrane, probed with the indicated antibodies, and visualized by ECL plus kit (GE Healthcare), in accordance with the manufacturers’ instructions. Rabbit polyclonal antibodies to RTEL1 were raised against a recombinant C-terminus fragment of human RTEL1 and affinity-purified, phospho-Chk2 (Thr68) and Chk2 antibodies were from Cell Signaling, TPP1 antibody from Bethyl Laboratories, POT1 antibody from Santa Cruz, FLAG M2 antibody or agarose beads and monoclonal -actin peroxidase conjugate were from Sigma-Aldrich. Rabbit antibodies to TRF1, TRF2, and hRap1 had been generated against recombinant proteins and affinity-purified. Immunoprecipitation. About 1 ?107 293 HEK cells overexpressing FLAGRTEL1 proteins or FLAG-GFP manage (as indicated) were lysed in 1 mL of RIPA buffer [1 Nonidet P-40, 1 Deoxycholate, 0.1 SDS, 150 mM NaCl, 10 mM Tris Cl, pH 7.5, 1 mM DTT, 1 mM PMSF, and 1?protease inhibitor mixtures (Sigma)] for 30 min at 4 . The lysates had been cleared by centrifugation for ten min at 20,000 ?g, and also the supernatants had been precleared with protein G Sepharose beads for 1 h at four . The precleared lysates were immunoprecipitated with FLAG agarose beads (Sigma) overnight at 4 , washed four occasions with RIPA buffer for ten min every single, and subjected to Western blot evaluation. Southern Blot Evaluation of Telomeric Restriction Fragments. Genomic DNA (two? g) was digested with AluI+MboI or AluI+HinfI restriction endonucleases, separated on a 0.7 agarose gel, denatured, and transferred to a Hybond N+ membrane (GE Healthcare). The blot was hybridized at 42 having a telomeric oligonucleotide probe, (TTAGGG)four or (TAACCC)4 5-end-labeled with T4 polynucleotide kinase (New England Biolabs) and 32P-ATP, and washed twice for 5 min with 0.2 M wash buffer [0.two M Na2HPO4 pH 7.two, 1 mM EDTA, and two (wt/vol) SDS] at area temperature and once with 0.1 M wash buffer at 50 , following Church and Gilbert (44), and exposed to an X-ray film or visualized by Typhoon 9410 Imager (GE Healthcare). Average telomere length was calculated by the pc program MATELO (45). Two-Dimensional Gel Electrophoresis. Two-dimensional gel electrophoresis was modified from ref. 46. Equal amounts of AluI+MboI digested DNA (ten?five g) was subjected to electrophoresis in a 0.four agarose gel (1st dimension) at area temperature and 30 V for 12?4 h, after which within a 1.2Deng et al.PNAS | Published on the web August 19, 2013 | EGENETICSPNAS PLUS(wt/vol) agarose gel (second dimension) containing 0.3 g/mL ethidium ALDH3 manufacturer bromide at four and 150 V for 6 h. The gel was processed as described above for the Southern analysis. In Fig. S5, two g of ligated DNA HindIII fragments had been electrophoresed collectively with all the digested genomic DNA in 2D gels and hybridized with DNA probes generated by random prime labeling of DNA HindIII fragments with 32P–dCTP. Metaphase Telomere FISH. LCLs had been subcultured into fresh medium and incubated at 37 for 24 h. Colcemid (0.1 g/mL; Gibco) was added for 4 h to accumulate mitotic cells. Cells had been collected by centrifugation at 112 ?g for ten min and suspended in 75 mM KCl hypotonic resolution at 37 for 25 min before fixation in fresh 3:1 methanol/acetic acid three to 4 occasions. Fixed cells were dropped onto cold and wet glass microscope slides and allowed to dry GPR139 Molecular Weight slowly inside a humid atmosphere. Metaphase chromosome spreads had been fixed in 4 (wt/.
