Characterization of a monoclonal antibody capable of reliably quantifying expression of Human Copper Transporter 1 (hCTR1)

Abstract

Human copper transporter 1 (hCTR1) is the high-affinity copper influx transporter in mammalian cells that also mediates the influx of cisplatin. Loss of hCTR1 expression has been implicated in the development of resistance to this cancer chemotherapeutic agent. It has turned out to be very difficult to develop antibodies to hCTR1 and polyclonal antibodies produced by different laboratories have yielded conflicting results. We have characterized a newly-available rabbit monoclonal antibody that reacts with an epitope on the N-terminal end of hCTR1 that now permits rigorous identification and quantification of hCTR1 using Western blot analysis. Postnuclear membrane (PNM) preparations made from cells engineered to express high levels of myc-tagged hCTR1, and cells in which the expression of hCTR1 was knocked down, were used to characterize the antibody. The identity of the bands detected was confirmed by immunoprecipitation, surface biotinylation and deglycosylation of myc-tagged hCTR1. Despite the specificity expected of a monoclonal antibody, the anti-hCTR1 detected a variety of bands in whole cell lysates (WCL), which made it difficult to quantify hCTR1. This problem was overcome by isolating post-nuclear membranes and using these for further analysis. Three bands were identified using this antibody in PNM preparations that migrated at 28, 33–35 and 62–64 kDa. Multiple lines of evidence presented here suggest that the 33–35 and 62–64 kDa bands are hCTR1 whereas the 28 kDa band is a cross-reacting protein of unknown identify. The 33–35 kDa band is consistent with the expected MW of the glycosylated hCTR1 monomer. This analysis now permits rigorous identification and quantification of hCTR1.

Molecular modulation of the copper and cisplatin transport function of CTR1 and its interaction with IRS-4

“…3A shows the effect of a 30 min exposure to 100 μM Cu on plasma membrane CTR1 in the wild type and the Y103A, C189S and K178A/K179A variants. Using a rabbit monoclonal antibody that we have previously extensively characterized [33], CTR1 was detected as bands of 33–35 and 62–64 kDa. The Cu exposure reduced CTR1 in the plasma membrane of the HEK293/myc-CTR1 WT cells to 15.9 ± 2.2% of that in the untreated cells ( p = 0.0007), whereas it reduced it to 67.0 ± 6.2% ( p = 0.07) of that in the untreated HEK293/myc-CTR1 Y103A cells, to 52.6 ± 3.6% ( p = 0.004) of that in the untreated HEK293/myc-CTR1 C189S cells and to 34.0 ± 9.0% ( p = 0.051) of that in the untreated HEK293/myc-CTR1 K178A/K179A cells as determined by quantitative imaging of 5 independent Western blot analyses normalized for Na/K ATPase content (Fig.…”

“…Protein concentrations were determined by detergent-compatible protein assay kit, DC ™ Protein Assay (Bio-Rad, Hercules, CA) and equal amount of proteins were incubated overnight at 4 °C with 100 μL streptavadin-coated beads (Thermo Scientific, Pittsburg, PA). The beads were collected and washed, after which the proteins bound to the beads were cleaved with 5% β-mercaptoethanol in 2 × SDS electrophoresis sample buffer and subjected to Western blot analysis using an antibody to the myc tag or to CTR1 [33] and an antibody to the Na/K ATPase that was used as a lane loading control. Western blot analysis was performed as previously described [31].…”

Dextran-Catechin: An anticancer chemically-modified natural compound targeting copper that attenuates neuroblastoma growth

“…Membranes were incubated with 10% skim milk powder in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 60 min, prior to washing once with TBST and incubating with antibodies against CTR1 [46] (rabbit anti-SLC31A1, ab129067, Abcam, Melbourne, VIC, Australia), PARP (rabbit anti-PARP antibody 9542, Cell Signaling Technology, Danvers MA, USA), or GAPDH (mouse anti-GAPDH, Abcam, Cambridge, UK). Membranes were washed and incubated with anti-rabbit or anti-mouse dilution of horseradish peroxidase-conjugated anti-antibody for 60 min.…”

Dextran-Catechin inhibits angiogenesis by disrupting copper homeostasis in endothelial cells

“…Whole cell extracts were separated by SDS-PAGE (12% poly-acrylamide gels) and transferred to a PVDF membrane using a transfer apparatus according to the manufacturer’s protocols (Bio-Rad). After incubation with 10% skim milk powder in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 60 min, the membrane was washed once with TBST and incubated with antibodies against CTR-1 28 (1:500 dilution; rabbit anti-CTR-1, ab129067, Abcam, Melbourne, VIC, Australia), ATOX-1 (1:500 dilution; rabbit anti-ATOX-1, ab154179, Abcam, Melbourne, VIC, Australia), VEGF-R2 (1:1000 dilution; rabbit anti-VEGF-R2, 2479, Cell Signaling Technology, Danvers MA, USA), or GAPDH (1:10,000; mouse anti-GAPDH, Abcam, Cambridge, UK). Incubation with primary antibodies for CTR-1, ATOX-1 and VEGF-R2 was at 4 °C overnight and at room temperature for 1 h for GAPDH primary antibody.…”