TAF6 was detected by immunocytochemistry using monoclonal antibodies that recognize an epitope present in all of the known isoforms of TAF6

June 1, 2017

s surrounding exon 7, which codes for amino acids 134185 of the PME-1 gene. The 59 and 39 homologous recombination fragments were subcloned into the NotI and BamHI sites and XhoI and internal HindIII sites of the pKO scrambler NTKV-1901 vector. Primers for 59 homologous end: 59-GC GCGGCCGC CAC TGG CAG ACA CTC TCT CGC-39 and 59-GC GGATCC CTC ACA GCT ATC TCC TTT ACC-39. Primers for the 39 homologous PP2A Activity Assays Phosphopeptide substrate assay. Phosphatase activity of E18 tissue homogenates was determined using the phosphopeptide KRpTIRR as a substrate with a PP2A immunoprecipitation phosphatase assay kit following the manufacturers recommendations. Free PO422 generated was quantified by measurement of the absorbance at 595 nm after addition of malachite green-molibdate reagent. A standard curve with free phosphate was used to determine the amount of phosphate generated. p-Nitrophenylphosphate assay. Phosphatase Role of PME-1 in PP2A Function activity was determined following similar protocols to those previously described. PP2A was immunoprecipitated with a PP2A antibody and KPT-9274 protein A-agarose beads from 2 mg of E18 tissue homogenates. Washed beads were resuspended in assay buffer and incubated in presence of 10 mM pNPP at 37uC for 20 min. The reaction was terminated by addition of an equal volume of Na2CO3 1.0 M and absorbance was measured at 405 nm. Phosphoproteomic analysis of mouse brain tissue E18 brains from either PME-1 or mice were homogenized in 10336422 Trizol and proteins isolated following the manufacturers directions. The protein pellet was reconstituted in 75 mL of 6M guanidineHCl with phosphatase inhibitors and solubilized at 60uC for 30 17876302 min. Debris was pelleted by centrifugation and protein concentration was measured in the supernatant using a Bradford-based protein assay. One milligram of total protein was brought to pH 8 by addition of 100 mM NH4HCO3, reduced with 10 mM dithiothreitol by incubation for 45 min at 60uC and alkylated with 20 mM iodoacetamide for 45 min in the dark. Proteomes were diluted with 100 mM NH4HCO3 to a final GuHCl concentration of 1 M and digested with trypsin overnight at 37uC. As an internal standard, the peptide FLApTGDGAR was added to each extract. Peptides were desalted using SPE columns, spiked with a second standard peptide, and concentrated by vacuum centrifugation to a volume of 50 mL. Reductive amination was performed as described, with modifications. Briefly, desalted peptides from PME-1 were added to 450 mL 1 M HEPES buffer, mixed with 40 mL formaldehyde, vortexed, and then mixed immediately with 40 mL of freshly prepared cyanoborohydride. The mixture was vortexed again and then allowed to react for 10 min. Tryptic peptides from PME-1 were labelled in a similar manner using d2-formaldehyde and sodium cyanoborodeuteride. Afterwards, peptides were desalted, combined and then subjected to strong cation exchange chromatography. SCX fractions containing phosphopeptides were then analyzed using an automated immobilized metal affinity chromatography -HPLC/MS platform coupled to an LTQ Orbitrap mass spectrometer. The mass spectrometer was operated in data dependent mode such that the top 5 most abundant ions in each MS scan were subjected to MS/MS. MS/MS were searched using the SEQUEST algorithm against a subdatabase of human, mouse and rat proteins derived from the NCBI non-redundant protein database. Postnatal Lethality in PME-1 Mice At the moment of birth, PME mice showed no obvious abnormali