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2 protocols using endothelin 3

1

Endothelin Receptor B Signaling Pathway

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Endothelin 3 was purchased from Sigma Aldrich and reconstituted to 1 mg/ml in water. siRNA specific for EDNRB (sc-39962), along with control siRNA (sc-37007) were purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA). EDNRB antibodies for Western blotting (ab117529) and flow cytometry (ab129102) and GAPDH antibody for Western blotting (ab9485) 31 (link) were purchased from Abcam (Cambridge, United Kingdom). pERK (#4370) and ERK (#4695) 32 (link) antibodies, along with pan AKT (4691), phospho AKT T308 (13038) and phospho-AKT1 S473 and phospho-AKT2 S474 (8599) were all purchased from Cell Signaling (Cell Signaling Technology, Beverly, MA). Secondary antibodies, including goat anti-mouse IgG-HRP, goat anti-rabbit IgG-HRP, goat anti-rabbit IgG-FITC were all purchased from Cell Signaling. Plasmid expression vectors encoding EDNRB-442 (RG 232943), EDNRB-536 (RC 216462), and EDNRB-436 (RC 225723) and accompanying empty vector controls (pCMV6-Entry vector) were purchased from Origene (Rockville, MD).
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2

Comprehensive Cell Culture Protocol

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RPMI culture medium was purchased from Corning (Corning, NY). Fetal bovine serum (FBS) was purchased from Sigma Chemical Co. (St. Louis, MO). Trypsin, pen/strep antibiotics, and puromycin were purchased from Gibco (Grand Island, NY). Trametinib (MEK inhibitor), Panobinostat (pan-HDAC inhibitor), Pictilisib (PI3K inhibitor), Bosentan Hydrate (EDNRB inhibitor), Verteporfin (YAP inhibitor), Entinostat (HDAC1/2/3 inhibitor), and Tubastatin A (HDAC 6 inhibitor) were purchased from Selleckchem (Houston, TX). PCI-34051 (HDAC8 inhibitor) was purchased from Cayman Chemical (Ann Arbor, MI). Endothelin-3 was purchased from Sigma Chemical Co. (St. Louis, MO). WNT5A was purchased from R&D Systems (Minneapolis, MN, USA). Antibodies for Western Blot and immunochemistry were purchased from Cell Signaling Technology (Danvers, MA), Sigma Chemical Co. (St. Louis, MO), Millipore (Bedford, MA) and Abcam (Cambridge, MA). The phospho-Receptor Tyrosine Kinase and phospho-Kinase array were purchased from R&D Systems (Minneapolis, MN, USA). Opti‐MEM medium, Lipofectamine 2000 and Live/Dead viability stain were purchased from Invitrogen/Life Technologies Corp). siRNA for ROR1/2, IGF-1R and YAP were purchased from Dharmacon RNA Technologies (Lafayette, CO). Nontargeting siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The Endothelin-3 Assay Kit was purchased from IBL (Takasaki, Japan).
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