Fig. 5: RGS6 interacts with SMAD4 and prevents complex formation between SMAD4 and phosphorylated-R-SMADs, slowing down nuclear entry of SMAD3 and SMAD4. | Cell Death & Disease

Fig. 5: RGS6 interacts with SMAD4 and prevents complex formation between SMAD4 and phosphorylated-R-SMADs, slowing down nuclear entry of SMAD3 and SMAD4.

From: RGS6 suppresses TGF-β-induced epithelial–mesenchymal transition in non-small cell lung cancers via a novel mechanism dependent on its interaction with SMAD4

Fig. 5

A RGS6-HA stable and control NSCLC cells were treated with TGF-β (5 ng/ml) for 24 h. Levels of phosphorylated R-SMADs and total levels of R-SMADs and SMAD4 were examined by western blotting. B Sample western blot with silver stain showing RGS6-associated proteins identified in mass spectrometry analysis. C HA-tagged RGS6 and Flag-tagged SMAD3 were co-transfected in A549 cells, cells were then treated with or without TGF-β (5 ng/ml) for 1 hour before subjected to immunoprecipitation with anti-HA antibody and probed for indicated proteins. D A549 cells were fixed and incubated with the indicated primary antibody simultaneously, then stained with a FITC-conjugated anti-mouse IgG (green, for RGS6) and a Cy3-conjugated anti-rabbit IgG (red, for SMAD4). Finally, nuclei were counterstained with DAPI (blue). Scale bar, 10 μm. E HA-tagged RGS6 and Flag-tagged SMAD3 were co-transfected in A549 cells, cells were then treated with or without TGF-β (5 ng/ml) for 1 hour before subjected to immunoprecipitation with anti-SMAD4 antibody and probed for indicated proteins. F, G RGS6-HA stable and control A549 cells were treated with or without TGF-β (5 ng/ml) for 2 h, followed by ICC staining for SMAD3 (F) and SMAD4 (G). DAPI staining was used for visualization of the nuclei. Scale bar, 20 μm. H RGS6-HA stable and control A549 cells were treated with or without TGF-β (5 ng/ml) for 1 hour followed by nuclear and cytoplasmic fractionation. Extracts of both fractions were subjected to immunoblotting for detection of indicated proteins. Lamin B1 and β-actin were used as internal controls for nuclear and cytoplasmic extracts, respectively. I RGS6-HA stable and control A549 cells were treated with TGF-β (5 ng/ml) for the indicated time periods, and subjected to western blotting for detection of indicated proteins. Left: a representative western blotting. Right, quantification of intensity of p-SMAD3 relative to total SMAD3 in three independent experiments using ImageJ software. J RGS6-HA stable and control A549 were treated with TGF-β (5 ng/ml) for 30 min, followed by TGF-β washout and simultaneous addition of 5 mM SB431542. After treatment of SB431542 for indicated time periods, cell lysates were collected and blotted for indicated proteins. Left: a representative western blotting. Right, quantification of intensity of p-SMAD3 relative to total SMAD3 in three independent experiments using ImageJ software.

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