Sumoylation directs NFATc1 into transcriptionally inactive
chromatin. A, 293T HEK cells were transfected with plasmids
encoding pHA-NFATc1/A, -c1/C, and -K349R/K702R/K914R and stimulated with
T/I+CaCl2 for 1 h. IF was performed to detect NFATc1 with anti-HA
and heterochromatin with anti-H3K9m3. Colocalization was analyzed by confocal
microscopy. The scale bar represents 10 μm. B, 293T HEK
cells were transfected with plasmids encoding SUMO1(S)-c1/A, -c1/C, and -
K349R/K702R/K914R, which were C-terminally fused to EYFP. Cells were
stimulated with T/I+CaCl2 for 1 h. IF was performed with
anti-H3K9m3, and colocalization of SUMO-fused NFATc1 (S-NFATc1) and
heterochromatin were analyzed by confocal microscopy. C, FLAG-SUMO1
was transfected along with c1/A, c1/C, and K349R/K702R/K914R plasmids,
C-terminally fused to EYFP into 293T HEK cells, and stimulated with
T/I+CaCl2 for 1 h followed by IF with anti-SUMO and anti-H3K9m3.
Tri-colocalization of NFATc1, SUMO1, and H3K9m3 was analyzed by confocal
microscopy. D, without sumoylation, NFATc1 colocalizes to hotspots of
transcription. Transfection and stimulation was as in C. To reveal
the colocalization pattern of NFATc1 and pol II-bodies, IF was performed with
anti-pol II antibodies. The scale bar represents 10 μm.
DAPI, 4′,6-diamidino-2-phenylindole.