MeCP2 were incubated in a reaction mixture with 40 mM Tris, pH
MeCP2 were incubated within a reaction mixture with 40 mM Tris, pH 7.5, ten mM MgCl2, 0.five mM CaCl2, 1 mM DTT, 50 g/mL calmodulin (Calbiochem), purified CaMKIV (recombinant, E. Coli, Life Technologies), 0.1 mM cold ATP, and 5 Ci (0.033 M) [-32P]-ATP (Perkin Elmer) within a 25 L reaction for 10 to 30 minutes at 30 . For in vitro kinase assays with PKA, purified MeCP2 variants had been incubated within a reaction mixture with 40 mM Tris, pH 7.5, 10 mM MgCl2, 1 mM DTT, PKA (catalytic subunit, mouse, recombinant, E. Coli, Calbiochem), 0.1 mM cold ATP, and 5 Ci (0.033 M) [-32P]-ATP within a 25 L reaction for 10 to 30 minutes at 30 .Nature. Author manuscript; readily available in PMC 2014 July 18.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptEbert et al.PageGeneration of anti-MeCP2 phospho-site-specific antibodies The polyclonal antibody that especially recognizes S86-phosphorylated MeCP2 was generated by injecting New Zealand White rabbits (Covance Research Goods) with all the peptide KQRR(pS)IIRDRGPM-C (Tufts Synthesis Facility, Boston, MA) conjugated to KLH. The antiserum was GLUT4 custom synthesis affinity-purified by incubation with a column that was conjugated with phosphorylated-S86 MeCP2 peptide, as well as the affinity-purified antibody was eluted. This eluate was then incubated with a column conjugated with unphosphorylated-S86 MeCP2 peptide, as well as the affinity-purified anti-MeCP2 pS86 antibody was collected inside the flow-through. The polyclonal antibody that particularly recognizes S274-phosphorylated MeCP2 was generated by injecting rabbits together with the peptide RKPG(pS)VVAAAAAEAKKKC conjugated to KLH. The antibody was affinity purified similar to the purification in the anti-MeCP2 pS86 antibodies. The polyclonal antibody that specifically recognizes T308phosphorylated MeCP2 was generated by injecting rabbits using the peptide CTVLPIKKRK(pT)RE conjugated to KLH. The antibody was purified over a column conjugated with MeCP2 T308 peptide, and also the affinity-purified anti-MeCP2 pT308 was eluted. The generation on the polyclonal rabbit antibody that particularly recognizes S421phosphorylated MeCP2 along with the polyclonal antibody that recognizes total MeCP2 irrespective of phosphorylation status have been previously described10. Stimulation of MeCP2 phosphorylation in cell culture and in vivoNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptCortical neuron cultures (E16 + 7 DIV) were membrane depolarized with 55 mM KCl by addition of 0.5 volumes of depolarization buffer (170 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and ten mM HEPES, pH 7.5). Alternatively, cultures have been treated with 20 M forskolin (Calbiochem) or 50 ng/mL BDNF (Peprotech) for 30 minutes or 1 hour. For bicuculline experiments, E16 + 14 DIV cortical neuron cultures had been treated with 20 M bicuculline (Sigma) for 30 to 120 minutes. For Western blot evaluation, cells were lysed in boiling sample buffer, to be able to preserve endogenous phosphorylation KDM3 review events and avert spurious phosphorylation events following cell lysis. Lysates have been boiled for ten minutes, passed via Wizard Minicolumns (Promega) to remove larger molecules and insoluble material, and resolved by 8 SDS-PAGE gels, normalized by cell number. Western blotting was performed with antibodies precise to MeCP2 phosphorylation sites (generated in our laboratory as described above) or particular to total MeCP2 (Men-8, Sigma) or beta-actin (ab8226, Abcam), all at 1:1000 dilutions. Western blotting was completed with HRPconjugated secondary antibodies an.