Western Blot Analysis of Copper Homeostasis Proteins

Cells were washed with ice cold PBS, then lysed in ice-cold RIPA buffer (50 mM Tris-HCl pH 8.0; 150 mM NaCl; 1 mM EDTA, 1% Triton X-100; 0.1% SDS; 0.5% sodium deoxycholate) supplemented with 1X Halt protease inhibitor cocktail (Thermo Fisher Scientific). Protein contents of the lysates were determined with the DC-Protein assay kit (Bio-Rad; Hercules, CA). Fifty micrograms of total protein was boiled with Laemmli dye for 5-10 minutes, then loaded on Tris-Glycine or Tris-Tricine gels and electrotransferred to low fluorescence PVDF membranes (EMD Millipore, Billerica, MA). Blots were stained with 1:1000 anti-CTR1 (gift from Dr. Marcus Kuo^{42 (link)}), 1:1000 mouse anti-CTR2 (National Cancer Institute^{43 (link)}), 1:1000 rabbit anti-ATOX1 (Abcam, Cabridge, MA) or 1:1000 rabbit anti-CCS (Santa Cruz Biotechnology, Dallas, TX) and 1:1000 mouse β-actin (Cell Signaling Technologies, Danvers, MA) primary antibodies overnight at 4°C. The blots were counter stained with 1:10,000 goat-anti rabbit 680LT and 1:5,000 goat-anti-mouse-800CW secondary antibodies (Li-Cor Biosciences, Lincoln, NE) and imaged with a Li-Cor Odyssey Imager (Li-Cor Biosciences). Images were quantified with Image J software (NIH, http://imagej.nih.gov/ij/).