NFATc1/C is sumoylated in vivo. A, NFATc1/C was
sumoylated. HA-tagged c1/C-coding vector was transfected into 293T HEK cells
either with FLAG-tagged SUMO1- or FLAG-expressing pcDNA, and cells were
treated with T/I for 6 h. Anti-FLAG immunoprecipitation was followed by IB
with anti-HA (arrows, SUMO-modified NFATc1/C) and whole cell lysates
(WCL) were analyzed for expression by anti-HA and anti-FLAG.
B, the SUMO site-deficient mutant K349R/K702R/K914R cannot be
sumoylated. Procedures were as in A, but K349R/K702R/K914R was
included, and cells were left unstimulated or treated for 6 h as indicated.
C, SUMO site mutants reveal site-specific sumoylation pattern of
c1/C. HA-tagged c1/C and different SUMO site mutants of c1/C were subjected to
IP as in A, and expression was compared by anti-HA (NFAT) and
anti-FLAG (SUMO) IB in whole cell lysates. D, NFATc1 precipitation
reveals sumoylated c1/C. Procedures were as in A, except
NFATc1/C-ER-stimulated by Tm + T/I for 4 h, and either anti-ER or anti-NFATc1
was used for IP and anti-SUMO1 for IB. E, in CD4+ T-cells
endogenous NFATc1 is sumoylated. Nuclei from CD4+ T-cells after 7
days restimulated or not by ionomycin or T/I for 5 h were subjected to IP and
IB as in D; IP-anti-NFATc1 + IB-anti-SUMO1
(arrow, sumoylated NFATc1/C, fourth lane, no antibody for
IP); reprobe of IB by anti-NFATc1, IB of nuclear lysates (NL) by
anti-SUMO1 and anti-NFATc1.