Journal: bioRxiv

Article Title: Isoform and protein region abnormalities of dysbindin and copper transporter proteins in postmortem schizophrenia substantia nigra

doi: 10.1101/343178

Figure Lengend Snippet: Schematic of copper transport between the blood and brain in controls (A) and schizophrenia (B). Stars show copper. CTR1, white arrows; ATP7A, black arrows; ATP7B, striped arrows; BBB, blood brain barrier; BCB, blood cerebrospinal barrier. Thinner arrows indicate decreased protein levels.

Article Snippet: The following antibodies and concentrations were used: rabbit anti-N-terminal ATP7A, 1:500, Novus Biologicals (NBP1-54906); rabbit anti-C-terminal ATP7A, 1:2000, Aviva Systems Biology (ARP33798_P050); rabbit anti-ATP7B, 1:1000, Abcam (ab135571); rabbit anti-transmembrane CTR1, 1:2,000, Novus Biologicals (NB100-402); rabbit anti-extracellular CTR1, 1:1000, Aviva Systems Biology (ARP43824_P050); rabbit anti-Dysbindin (targeting 1A and 1B/C), 1:2,000, Abcam (ab133652); and mouse anti-actin, 1:40,000, Millipore (MAB1501).

Techniques:

Journal: bioRxiv

Article Title: Isoform and protein region abnormalities of dysbindin and copper transporter proteins in postmortem schizophrenia substantia nigra

doi: 10.1101/343178

Figure Lengend Snippet: Protein levels of ATP7A (A-B), CTR1 (C-D), dysbindin 1A, 1B/C (E-F) and ATP7B (G-H) for analysis of diagnostic group and treatment status. Error bars represent standard deviation. Significant omnibus ANOVA results are: N-terminal ATP7A: p=0.02; C-terminal ATP7A: p=0.01; transmembrane CTR1: p=0.001. Significant ANOVAs were followed by post hoc tests illustrated by the following: *: p<0.05; **: p<0.01; ***: p<0.001.

Article Snippet: The following antibodies and concentrations were used: rabbit anti-N-terminal ATP7A, 1:500, Novus Biologicals (NBP1-54906); rabbit anti-C-terminal ATP7A, 1:2000, Aviva Systems Biology (ARP33798_P050); rabbit anti-ATP7B, 1:1000, Abcam (ab135571); rabbit anti-transmembrane CTR1, 1:2,000, Novus Biologicals (NB100-402); rabbit anti-extracellular CTR1, 1:1000, Aviva Systems Biology (ARP43824_P050); rabbit anti-Dysbindin (targeting 1A and 1B/C), 1:2,000, Abcam (ab133652); and mouse anti-actin, 1:40,000, Millipore (MAB1501).

Techniques: Diagnostic Assay, Standard Deviation

Journal: bioRxiv

Article Title: Isoform and protein region abnormalities of dysbindin and copper transporter proteins in postmortem schizophrenia substantia nigra

doi: 10.1101/343178

Figure Lengend Snippet: Numerical labeling of amino acid ranges are shown for each protein segment. Dotted boxes indicate antibody-specific epitopes. A. ATP7A protein structure. N-terminal antibody specifically binds to 225-273aa; C-terminal antibody binds to 1403-1452aa. B. CTR1 protein structure. Extracellular CTR1 antibody binds to 19-68aa; transmembrane CTR1 antibody specifically binds to 140-190aa. C. Intracellular diagram of copper chaperones and enzymes and schizophrenia-related alterations. Copper enters neurons after transport from astrocytes via CTR1 and is immediately bound by MT/ GSH. MT/GSH then delivers copper to chaperone ATOX1 or the TGN where it is delivered to other metalloenzymes (e.g., SCO1 → COX) via ATP7A. The SN exhibits more ATOX1 than other brain regions. SCO1 is a copper-requiring enzyme involved in the last step of the electron transport chain of ATP synthesis. SCO1, MT/GSH, COX, ATP, ATP7A and CTR1 are all downregulated or altered in schizophrenia. Abbreviations: MT, metallothionein; GSH, glutathione; APD, antipsychotic drug; SN, substantia nigra; COX, cytochrome c oxidase. References: 1) The present paper; 2) ; 3) ; 4) ; 5) ; 6) ; ; 7) ; 8) ; 9) ; 10) ; and 11) .

Article Snippet: The following antibodies and concentrations were used: rabbit anti-N-terminal ATP7A, 1:500, Novus Biologicals (NBP1-54906); rabbit anti-C-terminal ATP7A, 1:2000, Aviva Systems Biology (ARP33798_P050); rabbit anti-ATP7B, 1:1000, Abcam (ab135571); rabbit anti-transmembrane CTR1, 1:2,000, Novus Biologicals (NB100-402); rabbit anti-extracellular CTR1, 1:1000, Aviva Systems Biology (ARP43824_P050); rabbit anti-Dysbindin (targeting 1A and 1B/C), 1:2,000, Abcam (ab133652); and mouse anti-actin, 1:40,000, Millipore (MAB1501).

Techniques: Labeling

Journal: Nature Communications

Article Title: Inflammation mobilizes copper metabolism to promote colon tumorigenesis via an IL-17-STEAP4-XIAP axis

doi: 10.1038/s41467-020-14698-y

Figure Lengend Snippet: a Western blots analysis for copper trafficking-associated proteins from lysates of mouse colon epithelial organoids treated with different inflammatory cytokines for 8 h. Three independent experiments were done and representative blots were shown. Untr untreated. b IL-17 primed or unprimed mouse colon organoids were stained for STEAP4 (St4), E-cadherin (E-cad) and Ctr1 and integrin α. Scale bar, 100 µm. c SOD enzyme activity recovery from TTM inhibition in wild-type (WT) mouse organoids with or without 12 h of IL-17 priming. d SOD enzyme activity recovery in Steap4 wild-type (WT) and knockout (KO) organoids. e , f Fluorometric detection of intracellular copper in primary mouse colon organoids ( e ) or Ls174t cells ( f ). CF4 copper Fluor-4 probe, Ctrl CF4 control probe. Scale bar,100 µm. n = 5 biologically independent cell cultures. g Fluorometric detection of intracellular copper in Steap4 WT and Ctr1 knockdown cells. Cells were treated with or without IL-17 for 12 h and copper were supplemented into the medium for 6 h in all the groups. Scale bar,100 µm. n = 5 biologically independent cell cultures. h Atomic absorption spectroscopy measurement of copper content in Steap4 WT and KO Ls174t cells after copper addition. Cells were primed with or without IL-17 for 12 h and then 10 µM Cu(II) were supplemented into the medium of the indicated group for 6 h. n = 5 biologically independent cell cultures. i Copper content in cytosolic and mitochondrial fraction in Steap4 WT and KO cells, respectively. Cells were treated the same as described in panel ( h ). n = 5 biologically independent cell cultures. Results were normalized to cell numbers. All data show mean ± SEM. Two-tailed Student’s t test was performed for panel e (** P = 0.0057; *** P = 0.0004; **** P < 0.0001), f (*** P = 0.0004; **** P < 0.0001), g (* P = 0.015; ** P = 0.0027; *** P = 0.0004), h (* P = 0.0383; ** P = 0.0022; *** P = 0.0009), i (*** P = 0.0003; **** P < 0.0001; n.s., not significant). Data represent three independent experiments with similar results.

Article Snippet: The following primary antibodies were used for staining presented in the study: Ki67 (Cell Signaling Technology 12202, 1:200), Cleaved Caspase 3 (Cell Signaling Technology 9661, 1:300), STEAP4 (Proteintech, 11944-1-AP,1:100), IL-17A (R&D Systems, MAB317, 1:300), DYKDDDDK (Cell Signaling, 14793S, 1:300), Ctr1 (Novus Biologicals NBP2-36573, 1:100), TUNEL assay (Sigma, 11684795910).

Techniques: Western Blot, Staining, Activity Assay, Inhibition, Knock-Out, Control, Knockdown, Atomic Absorption Spectroscopy, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Theaflavin-3,3′-Digallate Enhances the Inhibitory Effect of Cisplatin by Regulating the Copper Transporter 1 and Glutathione in Human Ovarian Cancer Cells

doi: 10.3390/ijms19010117

Figure Lengend Snippet: Treatment with TF3 potentiated inhibitory effect of cisplatin against ovarian cancer A2780/CP70 and OVCAR3 cells via upregulating CTR1 protein expression. ( A ) The effect of TF3 at the designated concentrations on the protein levels of MRP2, ATP7A, ATP7B and CTR1 in ovarian cancer cells; ( B ) treatment with 7.5 μM TF3 upregulated CTR1 protein levels in ovarian cancer cells pretreated with 7.5 μM cisplatin; ( C ) transfection with CTR1 siRNA decreased CTR1 protein levels in ovarian cancer cells; ( D ) transfection with CTR1 siRNA enhanced the resistance of ovarian cancer cells to 7.5 μM cisplatin. Results are expressed as mean ± SD from three independent experiments. Significant differences among different treatments are marked with different letters ( p < 0.05), * ( p < 0.05) and ** ( p < 0.01).

Article Snippet: CTR1 siRNA and control siRNA were purchased from Santa Cruz Biotechnology (Danvers, MA, USA).

Techniques: Expressing, Transfection