Ir680

Cell lysates were prepared in RIPA buffer [20 mM Tris–HCl, pH 7.5, 0.1% (w/v) sodium lauryl sulfate, 0.5% (w/v) sodium deoxycholate, 135 mM NaCl, 1% (v/v) Triton X-100, 10% (v/v) glycerol, 2 mM EDTA], supplemented with Complete protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN, USA), and phosphatase inhibitors sodium fluoride (NaF) (20 mM) and sodium vanadate (Na₃VO₄) (0.27 mM). Western blot analysis was performed as previously described [28] (link). The total protein concentration was determined using the protein assay dye reagent from Bio-Rad Laboratories (Hercules, CA, USA). Cell lysates in SDS-sample buffer were boiled for 5 min and equal amounts of total protein analyzed by 10% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and western blotting. The antibodies used in this study are: rabbit monoclonal TRPM7 (at a 1:1000 dilution from Epitomics, Inc. (Burlingame, CA, USA)), rabbit polyclonal MycN (at a 1:500 dilution), goat polyclonal ODC (at a 1:500 dilution) and mouse monoclonal GADPH (at a 1:1000 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Secondary anti-mouse (at a 1:10,000 dilution) IR-680 or IR-800 (LI-COR Biosciences, Lincoln, NB, USA). Proteins were detected using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NB, USA) and analyzed with Licor Image Studio 2.0 acquisition and analysis software.