HTRF assay compared to Western Blot using phospho-SMAD3 assay

Cells were cultured to 80% confluency and treated with TGF-ß1 for 30 minutess. Following lysis, soluble supernatants were collected via centrifugation. Serial dilutions of ysates were performed and transferred into a low volume white microplate before finally adding HTRF phospho-SMAD3 detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot. The results reveal that the HTRF phospho-SMAD3 assay is 16-fold more sensitive than the Western Blot.

 

Validation on the mouse cell lines NIH/3T3 and C2C12

Mouse NIH/3T3 and C2C12 lines were plated (100,000 cells/well) and cultured 2h in complete culture medium, 37°C - 5% CO2. The day after, the cells were treated with increasing concentrations of TGF-ß1 for 30 minutes at 37°C - 5% CO2. After medium removal, the cells were lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred twice over into a low volume white microplate before adding 4 µL of the HTRF phospho-SMAD3 or total SMAD3 detection antibodies. The HTRF signal was recorded after an overnight incubation. In both lines, TGF-ß1 promotes the activation of SMAD3 by phosphorylation on Ser423/425, whereas the expression level of the protein remains fairly stable.

 

Overactivation of TGF-ß/SMAD signaling in hepatocytes and HSCs promotes liver fibrosis

Human LX-2* and HepG2 lines were plated (50,000 and 200,000 cells/well respectively) and cultured overnight in complete culture medium, 37°C - 5% CO2. Cells were then treated with increasing concentrations of TGF-ß1 for 30 minutes at 37°C - 5% CO2. After medium removal, cells were lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred twice over into a low volume white microplate before adding 4 µL of the HTRF phospho-SMAD3 or total SMAD3 detection antibodies. The HTRF signal was recorded after an overnight incubation. In both lines, TGF-ß1 treatment induces SMAD3 phosphorylation on Ser423/425, while the expression level of the protein is not significantly affected.

*LX-2 cell line provided by EMD Millipore (Part #SCC064)