Dorsomorphin dihydrochloride dorsomorphin

Recombinant human BMP2, DMH-1 (4-[6-[4-(1-methylethoxy) phenyl] pyrazolo [1,5-a]pyrimidin-3-yl]-quinoline), and dorsomorphin dihydrochloride (dorsomorphin) were obtained from R&D Systems (Minneapolis, MN, United States). SB431542 (catalog no. S4317) was purchased from the Sigma-Aldrich Corp. (St. Louis, MO, United States). A polyclonal goat anti-COX-1 (catalog no. sc-1752), polyclonal rabbit anti-SMAD1/5/8 (N-18; sc-6031-R), monoclonal mouse anti-α-tubulin (B-5-1-2; catalog no. sc-23948) and monoclonal mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (G-9; sc-365062) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, United States). A polyclonal rabbit anti-phospho-SMAD1 (Ser463/465)/SMAD5 (Ser463/465)/SMAD8 (Ser465/467) (D5B10) antibody was obtained from Cell Signaling Technology (Beverly, MA, United States). Horseradish peroxidase-conjugated rabbit anti-goat, goat anti-rabbit and goat anti-mouse secondary antibodies were obtained from Bio-Rad (Richmond, CA, United States).

The polyclonal anti-SMAD4 (9515) antibody was purchased from Cell Signaling Technology (Danvers, MA). Monoclonal anti-α-tubulin (sc-23,948) and polyclonal anti-CTGF (sc-14,939) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse and goat anti-rabbit immunoglobulin G were obtained from Bio-Rad Laboratories (Hercules, CA). HRP-conjugated donkey anti-goat immunoglobulin G was obtained from Santa Cruz Biotechnology. Recombinant human BMP2, dorsomorphin dihydrochloride (dorsomorphin), and dorsomorphin homolog 1 (DMH-1; 4-[6-[4-(1-methylethoxy) phenyl] pyrazole [1, 5-a] pyrimidin-3-yl]-quinoline) were obtained from R&D Systems (Minneapolis, MN). CTGF overexpression and CONTROL vectors were obtained from Gene Copoeia (Rockville, MD). In this study, two specific protein kinase inhibitors DMH-1 (an inhibitor of ALK2/3) and dorsomorphin (an inhibitor of ALK2/3/6) were used to conduct the loss of function experiment by blocking the downstream signaling pathways of ALK2/36. Specifically, the cells were pretreated with DMH-1 (0.25 μM) or dorsomorphin (10 μM) for 1 h, and cells were then treated with a vehicle control or 50 ng/ml BMP2 for 6 h (for examining mRNA levels) or 12 h (for examining protein levels).