Fig. 3: Ets-1 diminished the ubiquitination and proteasomal degradation of p-Smad3. | Cell Death & Disease

Fig. 3: Ets-1 diminished the ubiquitination and proteasomal degradation of p-Smad3.

From: Ets-1 deficiency alleviates nonalcoholic steatohepatitis via weakening TGF-β1 signaling-mediated hepatocyte apoptosis

Fig. 3

a Primary hepatocytes were stained with Hoechst in blue, anti-Ets-1 antibody in red and anti-Smad3 antibody in green, followed by assessment using confocal microscopy. Scale bar: 5 μm. b, c Hepatocytes were treated with TGF-β1 (10 ng/mL) for 6 h. b Cell lysates were immunoprecipitated with an anti-Smad3 antibody, followed by immunoblotting with an anti-Smad3 or anti-Ets-1 antibody. c Total lysates of cells were subjected to co-IP analysis with an anti-phosphorylated Smad3 (p-Smad3) antibody and immunoblot of Ets-1 or p-Smad3. d, e Hepatocytes were transfected with shEts-1 adenovirus (d) or co-transfected with HA-ubiquitin, Smad3 and Ets-1 plasmids (e) for 24 h and then treated with TGF-β1 for another 2 h. Finally, MG132 (25 μM) was added for 4 h. The lysates were used for immunoprecipitation with p-Smad3, and immunoblot was examined using p-Smad3, anti-ubiquitin (d) or anti-HA (e) antibody. f Hepatocytes were infected with shNC and the shEts-1 adenovirus for 24 h and treated with TGF-β1 (10 ng/mL) for 2 h. Cycloheximide (CHX, 50 ng/mL) was added, and the cells were harvested for 3, 6 and 9 h. Immunoblotting for p-Smad3 and Smad3 were performed. Quantitative data represent mean ± SEM. *P < 0.05 and **P < 0.01

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