Skip to main content
. 2020 Jun 25;39(32):5455–5467. doi: 10.1038/s41388-020-1376-3

Fig. 4. Biochemical and functional characteristics of neoGATA3 differ from wtGATA3.

Fig. 4

a Western blot showing the expression of wtGATA3 or neoGATA3 upon gene transduction in the GATA3-negative BT20 cells, after treatment with 50 µg/ml cycloheximide (CHX) for the indicated time. Vinculin was used as loading control. Quantification of relative band intensity is shown at the bottom. Images are representative of at least three independent experiments. b Immunofluorescence using the N-ter GATA3 antibody (top panels) or tag-specific antibodies (bottom panels, left: Flag, right: HA) in BT20 cells expressing either Flag-wtG3 or HA-neoG3, as indicated. DAPI was used to counterstain nuclei, GFP was expressed by the lentiviral vector used for the transduction. c EMSA assay performed with recombinant wtGATA3 or neoGATA3 and DNA fragment containing two GATAA motifs. d Reporter assay using the promoter regions of either CDH1 or CDH3 upstream of the luciferase cDNA. HEK293 cells were transiently transfected with the indicated constructs and luciferase activity was measured after 48 h. A GFP-expressing plasmid was co-transfected to normalize for transfection efficiency by western blotting (not shown). e Growth curve (left) and percentage of BrdU+ cells (right) measured in BT20 cells transduced with the indicated constructs. Data are represented as mean ± standard deviation of at least three independent experiments. Two-sided Student’s t test *P < 0.05, **P < 0.01.