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

Figure 3.

Figure 3

Role of noncanonical NFκB2 activation in enhanced GITR expression by TRAF3-deficient T cells.A, WT or Traf3−/− 2B4 T cells were stimulated via GITR as described in Experimental procedures, for the indicated times. Whole cell lysates were subjected to Western blot analysis for the indicated proteins, as described in Experimental procedures. Quantification (n = 3) of band intensity of p52 normalized to loading control (βactin) is shown on the right. B, total splenic T cells from the indicated mouse strains were stimulated via GITR as in (A), for the indicated times. Protein levels of p100 and p52 in whole cell lysates were determined by Western blot. Quantification (n = 5) of band intensity of p52 that is normalized to loading control (GAPDH) is shown on the right. C, inhibition of constitutive NFκB2 p100 processing. WT or Traf3−/− 2B4 cells were incubated for 2 h with 10 μM NIK-SMI, a small-molecule inhibitor of NIK kinase, prior to processing for Western blotting as described above. Quantification (n = 3) of band intensity of p52 normalized to loading control (GAPDH) is shown on the right. For A–C, ∗∗p < 0.01, and ∗p < 0.05 by unpaired t test. D, GITR mRNA levels of cells treated as in (C). In D, error bars represent mean ± SEM of values of one representative of three experiments. y-axis shows 2−ΔΔCt value of GITR mRNA as normalized to GAPDH. ∗∗p < 0.01 by unpaired t test. N = 12. E and F, inhibition of enhanced cell surface expression of GITR in TRAF3-deficient 2B4 T cells by NIK-SMI. WT or TRAF3-deficient 2B4 cells were treated with 10 μM NIK SMI for 2 h, prior to staining for GITR and analysis by flow cytometry. MFI ± SEM (n = 3) for GITR is shown in (F). A representative experiment is shown in (E). ∗∗p < 0.01 by unpaired t test.