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2 protocols using anti ctr1

1

Western Blot Analysis of Copper Homeostasis Proteins

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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 Kuo42 (link)), 1:1000 mouse anti-CTR2 (National Cancer Institute43 (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/).
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2

Quantitative Western Blot Analysis of CTR1 Protein

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Western blotting results were quantified using Image J software. Proteins were harvested from A549, H460, H1299 and A549/cDDP cells. Cells lysed in RIPA buffer containing PMSF (protease and phosphatase inhibitors) were quantified via BCA protein assay. Proteins separated on 10% SDS-PAGE (Invitrogen) were transferred onto PVDF membranes (PolyVinylidene Fluoride). After blocking in 5% defatted milk, membranes were incubated with primary antibodies overnight at 4°C. Membranes were washed with TBST and incubated with Horse Radish Peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Primary antibodies included: anti-CTR1 (1:1000, Abcam, Cambridge, Britain), anti-β-actin (1:1000, BOSTER, Wuhan, China) and anti-β-tublin (1:1000, BOSTER, Wuhan, China). Secondary antibodies included: HRP-Conjugated AffiniPure Goat Anti-Rabbit IgG (1:2000, ZSGB-BIO, Beijing, China) and HRP-Conjugated AffiniPure Goat Anti-Mouse IgG (1:2000, ZSGB-BIO, Beijing, China). Chemiluminesence western blotting reagents (Cell Signaling Technology, Danvers, MA, USA) were used to detect immunoreactive proteins. Protein bands were measured using Eagle Eye II software.
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