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. 2021 Aug 18;297(3):101097. doi: 10.1016/j.jbc.2021.101097

Figure 7.

Figure 7

Association of GITR, TRAF3, and TRAF2 in T cells.A, WT or Traf3−/− 2B4 cells were stimulated via anti-GITR Ab-conjugated protein G beads for times indicated, after which GITR signaling complexes were immunoprecipitated. C = control IP, where protein G beads conjugated with only the secondary anti-rat IgG Ab were used to IP GITR in unstimulated 2B4 cells, as described in Experimental procedures. IP samples were subjected to SDS-PAGE, and association of TRAF2 and TRAF3 with GITR was assessed by Western blot. Quantification (n = 3) of normalized band intensity of each protein is shown to the right of a representative figure. B, HA-tagged TRAF3 constructs used to transfect HEK293 cells for structure–function analysis. Zn Ring, Zn fingers, TRAF-N (N), and TRAFC (C) domains are shown. C, HEK293 T cells were transiently transfected with plasmids of the following combinations: Flag-tagged GITR plasmid alone (0) or + (1) WT TRAF3 (2), ΔN TRAF3, or (3) ΔC TRAF3. Cell lysates were immunoprecipitated with Abs to HA (TRAF3) or Flag (GITR), and association of GITR with TRAF3 mutants was assessed by Western blot. Protein expression in input lysates (each is 6% of total lysate) is also shown. Quantification of band intensity of indicated proteins is shown to the right of a representative figure. N = 3 for TRAF3:HA IP; N = 4 for GITR:flag IP. For both A and C, ∗∗p < 0.01, and ∗p < 0.05 by unpaired t test.

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