Figure 3.
Fig. 3. The VIVIT peptide selectively inhibits NFAT activation
but not calcineurin activity. (A) The VIVIT peptide did not
inhibit calcineurin phosphatase activity, assayed as radiolabel
released (counts per minute × 10^ 3)
from ^32P-phospho-RII peptide (9). The numbers next to each
peptide label indicate peptide concentrations (micromolar).
CsA/CypA complexes were used at 10 µM. (B) Selective
inhibition of NFAT reporter activity (11). Jurkat cells were
cotransfected with 3xNFAT-Luc (left panel) or 2xNF- B-Luc
(right panel) reporter plasmid, and with GFP and GFP-VIVIT
expression plasmids as indicated (measured in micrograms of
plasmid per 10^6 cells). Twenty-four hours after transfection,
cells were left untreated (open bars) or were stimulated for 6
hours with PMA and ionomycin (solid bars). (C) Calcineurin
dependence of NFAT and NF- B reporter
activity in T cells (11). Jurkat cells were transfected with
3xNFAT-Luc (left panel) or 2xNF- B-Luc
(right panel) reporter plasmid. Twenty-four hours after
transfection, cells were left unstimulated or were stimulated
for 6 hours with PMA and ionomycin (P+I) in the absence or
presence of CsA. (D) Inhibition of NFAT-dependent activation of
the IL-2 and TNF- promoters
(11). Jurkat cells were cotransfected with GFP or GFP-VIVIT
expression plasmids and with luciferase reporter plasmids driven
either by the human IL-2 promoter (left panel) or by the human
TNF- promoter
(right panel). Twenty-four hours after transfection, cells were
left unstimulated (open bars) or were stimulated for 6 hours
with PMA and ionomycin (solid bars) or with anti-CD3 and
anti-CD28 (hatched bars).
3)
from ^32P-phospho-RII peptide (9). The numbers next to each
peptide label indicate peptide concentrations (micromolar).
CsA/CypA complexes were used at 10 µM. (B) Selective
inhibition of NFAT reporter activity (11). Jurkat cells were
cotransfected with 3xNFAT-Luc (left panel) or 2xNF- 
B-Luc
(right panel) reporter plasmid, and with GFP and GFP-VIVIT
expression plasmids as indicated (measured in micrograms of
plasmid per 10^6 cells). Twenty-four hours after transfection,
cells were left untreated (open bars) or were stimulated for 6
hours with PMA and ionomycin (solid bars). (C) Calcineurin
dependence of NFAT and NF- 
promoters
(11). Jurkat cells were cotransfected with GFP or GFP-VIVIT
expression plasmids and with luciferase reporter plasmids driven
either by the human IL-2 promoter (left panel) or by the human
TNF-