Fig. 5

GADD45A suppressed LKB1 expression by interacting with Stat1. A Analysis of the GADD45A-associated proteins through mass spectrometry (MS) data. B Stat1 binding site prediction in the LKB1 promoter region using JASPAR. C Co-IP analysis of the binding between exogenous GADD45A and exogenous Stat1 in HEK293T cells co-transfected with Flag-GADD45A plasmid and HA-Stat1 plasmids. D Endogenously expressed GADD45A interacts with Stat1. Cell lysates from differentiated white adipocytes were IP with GADD45A or Stat1 antibodies and blotted with these antibodies. E Promoter activity of Lkb1 upon overexpression of GADD45A and/or Stat1 was detected by luciferase reporter assay. HEK293T cells seeded overnight in 24-well plates were co-transfected with pGL3-Lkb1, pCDNA3.1, Stat1, and varying amounts of GADD45A plasmids. The luciferase activities were examined after 24 h (n = 3). F Confocal immunofluorescence imaging of the subcellular localization of Stat1 (Red) and GADD45A (Green) in HEK293T cells 24 h after transfection with vectors expressing both proteins. Nuclei were stained with DAPI (blue). G Examination of the interaction of GADD45A with Stat1 protein by colocalization analysis. HEK293T cells were co-transfected with Stat1 and GADD45A plasmids, and 24 h later, cells were fixed with 4% (v/v) PFA, stained with DAPI and anti-Stat1 antibody, and analyzed by confocal microscopy. The intensity profiles of the indicated proteins along the plotted lines, as analyzed by ImageJ line scan analysis. Error bars represent SEM, *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Student’s t-test. Scale bars: 10 μm