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: Muscle & nerve

Article Title: Amyotrophic Lateral Sclerosis and swim training affect copper metabolism in skeletal muscle in a mouse model of disease.

doi: 10.1002/mus.28237

Figure Lengend Snippet: FIGURE 2 Effects of amyotrophic lateral sclerosis (ALS) disease progression and swim training on ATP7a, CTR1, and DMT1 protein level in skeletal muscle. ATP7a (A), CTR1 (B), and DMT1 (C) levels were measured in mice skeletal muscle. The cropped blots were used in the figure. Full-length blots are shown in Figures S2–S4. There were significant differences between the groups: ap < .05, bp < .01, dp < .001 versus ALS BEFORE group, Ap < .05, Bp < .01 versus ALS ONSET untrained group of mice, **p < .01, ***p < .001 versus wild type (WT) group of mice (Tukey's post hoc test), #p < .05, ##p < .01 between the indicated groups (Student t-test). The data are presented as the means ± SD (n = 6 in each group).

Article Snippet: Next, the membranes were rinsed three times in 1 TBST for 5 min, incubated with primary antibodies dissolved in blocking buffer with gentle shaking, and left overnight at 4 C. The following rabbit polyclonal antibody was used: CTR1/ SLC31A1 (Cat. No. 13086, Cell Signaling, Danvers, MA, USA, 1:1000), DMT1/anti-SLC11A1 (Cat. No. FNab07905, Fine Test, Wuhan, China, 1:500), anti-ATP7a (Cat. No. ab 125,137, Abcam, Cambridge, UK, 1:1000), and rabbit monoclonal antibody; CCS/CCS antibody (Cat. No. ab137131, Abcam, Cambridge, UK, 1:1000).

Techniques: Biomarker Discovery

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

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