L medium with B27 supplement (Invitrogen), 1 mMNature. Author manuscript; available in PMC 2014 July 18.Ebert et al.PageLglutamine, and one hundred U/mL penicillin/streptomycin. Cells were plated at 1 106 on 6well dishes, ten 106 on 10cm dishes, and 30 106 on 15cm dishes that had been pretreated with polyornithine. Phosphotryptic Mapping of MeCP2 Dissociated E16 mouse cortical neurons at six DIV were treated overnight with 1 M tetrodotoxin and 100 M APV (Tocris Bioscience) to cut down endogenous neuronal activity inside the culture. At 7 DIV, cortical neuron cultures, in 10cm dishes, have been labeled with two.five mCi of 32Porthophosphate (Perkin Elmer) for five hours. Neurons had been then left untreated or exposed to 55 mM KCl by addition of 0.five volumes of depolarization buffer (170 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and ten mM HEPES, pH 7.five) for ninety minutes, to induce membrane depolarization and robustly model neuronal activity. Neurons were washed once in PBS, lysed in TTN lysis buffer (30 mM Tris, pH 7.5, 1 M NaCl, 3 Triton X100, five mM EDTA, ten mM glycerolphosphate, ten mM NaF, two mM Na3VO4, and 1x comprehensive EDTAfree protease inhibitor cocktail [Roche]), and sheared using a 22gauge needle. Insoluble material was pelleted at 17,000 g and removed. Lysates have been diluted with equal volumes of H2O to lower NaCl concentration to 500 mM. Lysates had been immunoprecipitated with an antitotal MeCP2 antibody (antibody for the Cterminus of MeCP2 that was generated inhouse as described in10) bound to Protein A sepharose beads, for two hours whilst rotating at four . Immunoprecipitates was washed 4 times in TTN buffer (diluted to 500 mM NaCl and 1.five Triton X100) and resolved by SDSPAGE. The dried gel was exposed to autoradiography.Methyl 4-bromo-1H-pyrazole-3-carboxylate web Phosphotryptic mapping of MeCP2 followed the procedure detailed in25. The MeCP2 band was excised in the gel and digested with trypsin (TPCKtreated, Worthington). The tryptic phosphopeptides have been separated in twodimensions by thinlayer electrophoresis, employing pH 1.9 electrophoresis buffer (two.5 formic acid [88 w/v] and 7.8 glacial acetic acid), for 30 minutes at 1000 volts and by thinlayer chromatography, utilizing the phosphochromatography buffer (37.212127-83-8 Chemscene 5 nbutanol, 25 pyridine, and 7.PMID:23996047 5 glacial acetic acid), on glassbacked TLC plates (20 20 cm, one hundred uM cellulose, EM Science). The phosphotryptic maps were visualized by autoradiography. Experiments shown have been repeated higher than 3 occasions employing biological replicates. In Vitro Kinase Assays MeCP2 fragments had been generated by calcium phosphate transfection of HEK 293T cells with constructs expressing FLAGtagged Nterminal MeCP2 variants from amino acid 1 to 193 or Cterminal MeCP2 variants from amino acid 173 to 484. Missense mutations at putative internet sites of phosphorylation were generated by sitedirected mutagenesis utilizing Quickchange (Stratagene) and fully sequenced through the complete subcloned segment. HEK 293T cells have been transfected by calcium phosphate. Fortyeight hours immediately after transfection, the exogenous MeCP2 variants have been harvested in Lysis buffer (50 mM Tris, pH 7.5, 500 mM NaCl, 2.5 Triton X100, two mM EDTA, 10 mM NaF, 2 mM Na3VO4, 1 mM DTT, and 1x full EDTAfree protease inhibitor cocktail [Roche]), immunoprecipitated with antiFLAG antibodies (M2, Sigma), and eluted in the beads with FLAG peptides at 150 ng/L concentration. The purified MeCP2 variants have been phosphorylated applying in vitro kinase assays. For in vitro kinase assays with CaMKIV, Cterminal fragments of MeCP2 were incubated inside a reaction mixture w.