MDA-MB231 and PC-3 tumor cell lines and COS-7 immortalized cells were obtained from the American Type Culture Collection and cultured in a humidified incubator with 5% CO2 at 37°C in Dulbecco’s modified Eagle’s medium or RPMI 1640 media supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin, respectively. Antibodies directed against Sema4C (sc-136445), N-cadherin (sc-7939), fibronectin (sc-8422), Zeb1 (sc-25388), SMAD1 (sc-7965), ID1 (sc-488), actin (sc-1616), PlexinB2 (sc-34504), TGF-β–R2 (sc-17792), and ACTR1/ACVR1 (sc-374523) were all from Santa Cruz Biotechnology. Anti-vimentin (VIM-3B4) was from Merck. Anti–p-SMAD1/5/9 (D5B10, no. 13820) and anti–p-SMAD2/3 (D27F4, no. 8828) were from Cell Signaling Technology. Anti-vinculin (V4505) and anti-Flag (clone M2_F3165) were from Sigma-Aldrich. Anti–E-cadherin was from BD Transduction Laboratories (catalog no. 610182). Anti–TGF-β–R1 antibody was from Abcam (ab31013). Anti-BMPR1A was purchased from Proteintech (catalog no. 12702-1-AP). Cell treatments with 1 to 5 μM SB-431542 hydrate (Sigma-Aldrich) were performed for 48 to 72 hours, as described. Purified recombinant Sema4C ectodomain (catalog no. AF6125) was purchased from R&D Systems. A tumor multiple tissue array containing primary and metastatic breast cancer samples (BR1008A) was obtained from US Biomax Inc. (Rockville, MD).

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.