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2 protocols using dimethylthiourea

1

Protease Inhibitor Cocktail Preparation

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α1-antitrypsin, trypsin inhibitor from Glycine max (soybean), leupeptin, aprotinin were from Sigma-Aldrich, and dissolved in F12K medium without serum. GM6001 and E64 were from Santa Cruz Biotechnology, and dissolved in DMSO. Protease inhibitor cocktail solution in DMSO was from Sigma. It contained AEBSF (104 mM), aprotinin (80 μM), bestatin (4 mM), E64 (1.4 mM), leupeptin (2 mM) and pepstatin (1.5 mM). BMS200261 was from KeraFast (Boston, USA), and dissolved in water. N-acetylcysteine was from Sigma, and dissolved in cell culture medium without serum and pH adjusted to 7.0 with sodium hydroxide. Dimethylthiourea was from Acros, and dissolved in cell culture medium without serum. 1-(2-Cyano-3,12,28-trioxooleana-1,9(11)-dien-28-yl)-1H-imidazole (CDDOIm) was from the National Cancer Institute or Tocris, and dissolved in DMSO.
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2

Surfactant Protein Immunoblotting Protocol

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Cell culture media and cell culture reagents were from Life Technologies
(Carlsbad, CA). Polymyxin B, actinomycin D,
Nω-nitro-l-arginine methyl ester
(l-NAME), α1-antitrypsin and soybean trypsin inhibitor were from
Sigma-Aldrich (St. Louis, MO). Dimethylthiourea was from Acros Organics (Morris,
NJ). SP-A Abs were from Chemicon International (catalog # AB3030) (Temecula, CA)
or kindly provided by Joanna Floros, Penn State College of Medicine (Hershey,
PA). SP-B Abs were from Chemicon International (catalog # AB3034) (Temecula, CA)
or from Seven Hills Bioreagents (catalog # WRAB-48604) (Cincinnati, OH). SP-B
Abs from Chemicon International were reacted with the blot under reducing
conditions, whereas SP-B Abs from Seven Hills Bioreagents were reacted under
non-reducing conditions. SP-C (catalog # sc-13979) and actin (catalog # sc-1616,
sc-47778) Abs were from Santa Cruz Biotechnology (Dallas, TX) and tubulin Abs
(catalog # MS-581-P0) were from Thermo Fisher Scientific (Fremont, CA). Mouse
monoclonal SP-D Abs (catalog # WMAB-1A10A9) were from Seven Hills Bioreagents
(Cincinnati, OH). TTF-1 Abs were purified by Protein A-Sepharose chromatography
from rabbit antisera against N-terminal portion of rat T/EBP (TTF-1/Nkx2.1)
kindly provided by Shioko Kimura, National Cancer Institute (Bethesda, MD). We
have used these Abs for Western blot detection of TTF-1.23 (link)
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