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. 1999 Aug 17;96(17):9803–9808. doi: 10.1073/pnas.96.17.9803

Figure 1.

Figure 1

Targeted disruption of the murine traf5 gene. (A) A portion of the traf5 gene containing 5 exons (solid boxes) is indicated (Top). The targeting construct (Middle) was designed to replace exon II, which encodes the C-terminus of the RING finger domain with a pMC1neo cassette in a reverse orientation. (Bottom) The mutant locus resulting from homologous recombination. An extra EcoRI restriction site introduced by the pMC1neo cassette was used for the detection of homologous recombination. (B) Southern blot analysis of genomic DNA from wild-type and traf5+/− (clone 46, 48) ES cells. DNA was digested with EcoRI and hybridized with probe A as described in A. (C) PCR analysis of genomic DNA from wild-type, traf5+/−, and traf5−/− animals. Tail DNA was amplified by PCR using P1 and P2 primers as described in A. (D) Western blot analysis of TRAF5 protein expression. Total lung lysates from wild-type, traf5+/−, and traf5−/− mice were subjected to affinity purification by using glutathione beads preadsorbed with GST-LT-βR fusion protein (L) or control beads preadsorbed with GST protein alone (G). The positive control lysate (P) was prepared from 293 cells transfected with TRAF5 expression vector. The bound materials were fractionated by SDS/PAGE, and the Western blot was probed with polyclonal antibodies raised against a peptide derived from TRAF5-N, TRAF5-C, or N-terminal TRAF2 (25).