Fig. 1. Structure of the Tak1 genetrap allele and western analysis of TAK1 protein. (A) The genetrap insertion in the Tak1 locus. The insertion is between exon 1 (E1) and exon 2 (E2) of Tak1. The genetrap contains a splice acceptor (SA) upstream of the ßgeo gene, which is linked via an internal ribosome entry site (I) to the placental alkaline phosphatase gene (PLAP). Genomic DNA was digested with HindIII (Hd3) for Southern blotting using a probe just outside the insertion site (red box). (B) Genotyping by Southern blot and transcript levels in Tak1/ embryos. Southern blot of DNA extracted from yolk sacs of E9.5 embryos derived from Tak1+/ intercrosses. The wild-type and genetrap alleles result in probe hybridization to 2.7 kb and 6.7 kb bands, respectively. (C) rtPCR analysis of wild-type transcript (primers amplifying exon1 to exon 2 of Tak1), fusion transcript (primers amplifying exon1 to ßgeo) and actin as a control for the amount of cDNA that was amplified in each sample. Samples that did not contain reverse transcriptase (–RT) were used as a control for DNA contamination. A small amount of wild-type transcript is detected in Tak1/ samples. (D) Western blot analysis of lysates from E10.5 embryos derived from Tak1+/ intercrosses with a polyclonal antibody to full-length TAK1. A 70 kDa doublet is visible in Tak1+/+ and +/ samples, but is missing in the Tak1/ sample. The TAK1 antibody used also detects a GST background band (labeled GST) and a band of 150 kDa in Tak1+/ and / samples, which is probably the predicted fusion protein between exon 1 of Tak1 and ßgeo. The amount of total protein per lane was determined by assaying ß-tubulin levels.