Figure 7

FBXL19 regulates TGFβ1-induced E-cadherin down-regulation - A. OE19 cells were transfected with FBXL19-V5 plasmid (0–4 μg) or OE33 cells were transfected with FBXL19-V5 (4 μg) for 2 days, and then the cell lysates were analyzed by immunoblotting with Rac3, V5 tag and β-actin antibodies. Rac3 levels were quantified by Image J software. B. Cell lysates from FBXL19-V5-overexpressed HEK293 or OE19 cells were analyzed for Rac3 activity by using a PBD-GTPγ-Rac3 pull down assay. Total cell lysates were analyzed by immunoblotting with Rac3, V5, and β-actin antibodies. Activated Rac3 levels were quantified by Image J software. C. OE19 cells were transfected with FBXL19-V5 plasmid for 24 h followed by treatment of TGFβ1 (0–5 ng/ml) for 4 days. The cell lysates were analyzed by immunoblotting with E-cadherin, V5 tag, and β-actin antibodies. E-cadherin levels were quantified by Image J software. D. OE33 cells were transfected with FBXL19-V5 plasmid for 24 h followed by treatment of TGFβ1 (5 ng/ml) for 4 days. The cell lysates were analyzed by immunoblotting with E-cadherin, V5 tag, and β-actin antibodies. E-cadherin levels were quantified by Image J software. E. OE19 cells were transfected with Rac3-V5, FBXL19-V5, or Rac3-V5 + FBXL19-V5 plasmids for 48 h. Cell lysates were then analyzed by immunoblotting with Snail, V5 tag, and β-actin antibodies. Snail levels were quantified by Image J software. F. OE19 cells were transfected with FBXL19-V5 or vector plasmid for 24 h and then incubated with TGFβ1 (5 ng/ml) for 4 days. Cells were fixed and immunostained with E-cadherin (green). Nuclei were stained with DAPI (red). G. Cell length was measured by Image J software. All the blots and images are representative of three independent experiments.