Western Blot Analysis of BMP Signaling

The expression of BMPER, Collagen1 (Col1), Phospho-Smad1 (p-Smad1), Smad1, Phospho-Smad3 (p-Smad3) and Smad3 were evaluated by western blotting essentially as described33 (link). For western blotting analysis, the following antibodies were used: BMPER (ab75183, 1:1000, Abcam, Cambridge, MA, USA), Collagen1 (ab21285, 1:250, Abcam), p-Smad1 (5753s, 1:1000, Cell Signaling, Danvers, MA, USA), Smad1 (9743s, 1:1000, Cell Signaling), p-Smad3 (9520p, 1:1000, Cell Signaling), Smad3 (9523s, 1:1000, Cell Signaling) and β-actin (12620, 1:1000, Cell Signaling). The secondary antibodies used were Peroxidase AffiniPure F(ab’)2 Fragment Donkey Anti-Rabbit IgG (H+L) (Code: 711-036-152, 1:5000, Jackson ImmunoResearch, West Grove, PA, USA) and Peroxidase AffiniPure Goat Anti-Mouse IgG, Fcγ Subclass 1 Specific, (Code: 115-035-205, 1:5000, Jackson ImmunoResearch,). Pierce ECL Western Blotting Substrate (Cat. # 32106, Life Technologies, Grand Island, NY, USA) was used to visualize on a ChemiDoc MP Imaging System (Bio-RAD, Philadelphia, PA, USA). Quantitative densitometric analysis relative to β-actin was used to aid clarity.