Splice sites were determined by alignments to mm5. A total of 191 sequences for which the final splice site was more than 50 bases from the stop codon were flagged as putative NMD targets. A number of the final splice sites were suspected as artefactual alignments with very short pre dicted intron lengths. To selleck chemical Abiraterone remove these Inhibitors,Modulators,Libraries artefacts a further requirement was imposed that the minimum intron length had to be greater than 80 bases. This reduced the set to 120 predicted NMD candidates. These predictions were reviewed manually. All NMD predictions are provided in Additional data file 8 and online. Feature based assessment of protein function For each locus, InterProscan predictions were used to assess changes in domain content of each variant.

Using this we determined Inhibitors,Modulators,Libraries the domain content for each full length transcript and then used predictions for every transcript to determine the domain complement for each locus. Domain changes were assessed by comparing the domain content of the pre dicted peptide with the domain complement of the locus. Additionally, for the receptor set, TMHMM and signalP pre dictions were used to detect transmembrane domains and signal peptides. Variant receptors that lacked the transmembrane domain but retained the signal peptide were classified as probably secreted decoy receptors, whereas transmembrane Inhibitors,Modulators,Libraries forms lacking the catalytic domain were clas sified as probably tethered decoys. Subcellular localization of Csf1r variant transcripts cDNA clones of variant Csf1 receptor were subcloned into a mammalian expression vector.

HeLa cells were transiently transfected for 16 hours, formalin fixed, and Inhibitors,Modulators,Libraries processed for immunofluorescence. Recombinant Csf1r was detected using the rat monoclonal AFS98 antibody. Validation of Csf1r variant Inhibitors,Modulators,Libraries transcripts RNA was harvested from cells using the RNeasy kit. First strand synthesis was car ried out on 1g total RNA using Superscript III. Real time PCR was performed with the SYBR qPCR SuperMix UDG kit. Twenty microliter reactions were performed in an ABI 7700, with 35 cycles of 1 minute elongation at 60 C. all reactions were performed in duplicate. MLN2238 Relative fold change of full length and variant were calculated using the delta Ct method. 5 RACE experiments 5 RACE experiments were performed using an enzymatic oligo capping method that ensures capture of full length capped 5 ends. Reverse transcrip tion using random hexamers was carried out to generate 12 libraries from six tissues. Nested primers running back towards the 5 ends of the transcripts were then used in conjunction with a primer against the 5 ligated oligo to amplify the 5 ends of these cDNAs. The PCR products were then cloned into the pCR4 TOPO vector and 24 colonies from each library sequenced.