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. 1999 Feb 16;96(4):1421–1426. doi: 10.1073/pnas.96.4.1421

Figure 1.

Figure 1

Characterization of the relative binding affinities of TRAFs 2, 3, 5, and 6 for the CD40 cytoplasmic tail and determination of the minimal binding regions within the CD40 C17 region. (A) TRAFs 2 and 3 bind to the CD40ct with much greater affinity than TRAFs 5 and 6. The same amount of glutathione beads bound with the GST-mouse CD40ct fusion protein was incubated with extracts from 293T cells transfected with pEBB-Flu TRAF2, pEBB-Flu TRAF3, pEBB-Flu TRAF5, or pEBB-Flu TRAF6. The beads were washed, and bound proteins were detected by Western blot analysis with anti-Flu antibodies (Fig. 1A Upper). Equal amounts of extracts (5% of input used for GST pull-down) were analyzed by Western blot analysis with anti-Flu antibodies to ensure similar levels of expression of the different Flu-tagged TRAFs (Fig. 1A Lower). (B) The minimum binding regions of TRAF2 and TRAF3 within the C17 region are different. Lysates from 293T cells expressing Flu-TRAF2 or Flu-TRAF3 were incubated with GST, GST-C17, GST-C10N, or GST-C10C fusion proteins immobilized on glutathione beads. The bound proteins then were analyzed by Western blot analysis with anti-Flu antibodies.