Journal: PLoS ONE

Article Title: Novel ATPase Cu 2+ Transporting Beta Polypeptide Mutations in Chinese Families with Wilson's Disease

doi: 10.1371/journal.pone.0066526

Figure Lengend Snippet: Clinical data from the 20 patients with the ATP7B gene mutations.

Article Snippet: The blots were blocked for 1 h in 5% milk in TBST and incubated at 4°C overnight with rabbit anti- ATP7B polyclonal antibody (Novus Biologicals, Littleton, CO, USA), rabbit anti- minichromosome maintenance protein 7 (MCM7) monoclonal antibody (Millipore Biologicals, Billerica, MA, USA), rabbit anti- sterol regulatory element binding protein 1 (SREBP1) polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), mouse anti- B-cell CLL/lymphoma 2 (BCL2) polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), mouse anti- BCL2-associated X protein (BAX) polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) and mouse anti-β-actin polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA).

Techniques:

Journal: PLoS ONE

Article Title: Novel ATPase Cu 2+ Transporting Beta Polypeptide Mutations in Chinese Families with Wilson's Disease

doi: 10.1371/journal.pone.0066526

Figure Lengend Snippet: Statistical differences between ATP7B siRNA transfected groups and AllStars Negative Control siRNA groups are indicated as * ( P <0.01), or untreated group are indicated as # ( P <0.01).

Article Snippet: The blots were blocked for 1 h in 5% milk in TBST and incubated at 4°C overnight with rabbit anti- ATP7B polyclonal antibody (Novus Biologicals, Littleton, CO, USA), rabbit anti- minichromosome maintenance protein 7 (MCM7) monoclonal antibody (Millipore Biologicals, Billerica, MA, USA), rabbit anti- sterol regulatory element binding protein 1 (SREBP1) polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), mouse anti- B-cell CLL/lymphoma 2 (BCL2) polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), mouse anti- BCL2-associated X protein (BAX) polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) and mouse anti-β-actin polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA).

Techniques: Transfection, Negative Control

Journal: PLoS ONE

Article Title: Novel ATPase Cu 2+ Transporting Beta Polypeptide Mutations in Chinese Families with Wilson's Disease

doi: 10.1371/journal.pone.0066526

Figure Lengend Snippet: (A) Western blot analysis of ATP7B , BCL2, BAX, SREBP1 and MCM7 in HepG2 cells following treatment with targeted siRNA and AllStars Negative Control siRNA for 24, 48, and 72 h. β-actin was shown below as loading control. Protein levels of (B) ATP7B , (C) BCL2, (D) BAX, (E) SREBP1 and (F) MCM7 were quantified by densitometry and expression is shown as arbitrary units. Statistical differences for expression of the proteins between ATP7B siRNA transfected groups and AllStars Negative Control siRNA groups are indicated as * ( P <0.05), or ATP7B siRNA untreated group are indicated as # ( P <0.05).

Article Snippet: The blots were blocked for 1 h in 5% milk in TBST and incubated at 4°C overnight with rabbit anti- ATP7B polyclonal antibody (Novus Biologicals, Littleton, CO, USA), rabbit anti- minichromosome maintenance protein 7 (MCM7) monoclonal antibody (Millipore Biologicals, Billerica, MA, USA), rabbit anti- sterol regulatory element binding protein 1 (SREBP1) polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), mouse anti- B-cell CLL/lymphoma 2 (BCL2) polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), mouse anti- BCL2-associated X protein (BAX) polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) and mouse anti-β-actin polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA).

Techniques: Western Blot, Negative Control, Control, Expressing, Transfection

Journal: PLoS ONE

Article Title: Novel ATPase Cu 2+ Transporting Beta Polypeptide Mutations in Chinese Families with Wilson's Disease

doi: 10.1371/journal.pone.0066526

Figure Lengend Snippet: HepG2 cells were stained with 1 μM of Hoechst 33342. Cells were treated with siRNA for 24 h and followed by copper sulfate treatment for 24 h. (A) Treated with 5 nM AllStars Negative Control siRNA. (B) Treated with 5 nM ATP7B siRNA. (C) Treated with 5 nM AllStars Negative Control siRNA+ 50 μM copper sulfate . (D) Treated with 5 nM ATP7B siRNA+ 50 μM copper sulfate. (E) Treated with 5 nM AllStars Negative Control siRNA + 200 μM copper sulfate. (F) Treated with 5 nM ATP7B siRNA+ 200 μM copper sulfate.

Article Snippet: The blots were blocked for 1 h in 5% milk in TBST and incubated at 4°C overnight with rabbit anti- ATP7B polyclonal antibody (Novus Biologicals, Littleton, CO, USA), rabbit anti- minichromosome maintenance protein 7 (MCM7) monoclonal antibody (Millipore Biologicals, Billerica, MA, USA), rabbit anti- sterol regulatory element binding protein 1 (SREBP1) polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), mouse anti- B-cell CLL/lymphoma 2 (BCL2) polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), mouse anti- BCL2-associated X protein (BAX) polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) and mouse anti-β-actin polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA).

Techniques: Staining, Negative Control

Journal: Molecular Pain

Article Title: Differential expression of ATP7A, ATP7B and CTR1 in adult rat dorsal root ganglion tissue

doi: 10.1186/1744-8069-6-53

Figure Lengend Snippet: Copper transporter gene expression in rat tissues determined by quantitative PCR

Article Snippet: The slides were incubated with a chicken anti-ATP7A (1:1000; Abcam), a rabbit anti-ATP7B antibody (NB100-360, Novus Biologicals, Littleton, CO, USA) or a rabbit polyclonal anti-hCTR1 antibody (1:500, Novus Biologicals, Littleton, CO, USA), respectively, at room temperature overnight.

Techniques: Gene Expression

Journal: Molecular Pain

Article Title: Differential expression of ATP7A, ATP7B and CTR1 in adult rat dorsal root ganglion tissue

doi: 10.1186/1744-8069-6-53

Figure Lengend Snippet: ATP7A protein expression in rat DRG tissue . (A) Detection of ATP7A protein by Western blot analysis in DRG of rats aged 4 weeks (lane 1), 12 weeks (lane 2) and 20 weeks (lane 3). Beta actin was probed as a loading control. (B) a and b, neuronal ATP7A immunoreactivity in rat L5 DRG tissue associated with cell bodies of small neurons, intense punctuate vesicular structures in cytoplasm (solid arrows), diffuse cytoplasmic and granular membrane staining (broken arrows), without staining of nuclei (n) or other tissue elements. b was the enlarged frame in a. Inset in a was a negative control. c, Fluorescent immunohistochemistry shows little overlap between ATP7A-immunoreactive (IR) neurons (green), phosphorylated neurofilament heavy subunit (pNF-H)-IR neurons (red), DAPI-stained satellite cells (asterisk) or nerve fibres (f). Scale bar, 20 μm.

Article Snippet: The slides were incubated with a chicken anti-ATP7A (1:1000; Abcam), a rabbit anti-ATP7B antibody (NB100-360, Novus Biologicals, Littleton, CO, USA) or a rabbit polyclonal anti-hCTR1 antibody (1:500, Novus Biologicals, Littleton, CO, USA), respectively, at room temperature overnight.

Techniques: Expressing, Western Blot, Control, Membrane, Staining, Negative Control, Immunohistochemistry

Journal: Scientific Reports

Article Title: Novel and less invasive biomarker assays to measure liver ATP7B in Wilson disease patients

doi: 10.1038/s41598-025-21455-y

Figure Lengend Snippet: ATP7B RNA is detectable in hepatocytes in both fine needle aspiration (FNA) and core needle biopsy (CNB) samples. ( A ) Average relative expression of ATP7B mRNA for each CNB sample was calculated relative to the control samples using TF as the reference gene. ( B ) Average relative expression of ATP7B mRNA for each FNA sample was calculated relative to the average of control samples using TF as the reference gene. Each bar represents the mean of 3 technical replicates. Each dot represents a single technical replicate.

Article Snippet: Anti-ATP7B capture antibody (Abcam, UK, Cat# 131208), anti-ATP7B detection antibody (Novus Biologicals, USA, Cat# NB100-361) and ATP7B recombinant protein (Origene, USA, Cat# TP323635) were purchased from commercial sources.

Techniques: Expressing, Control

Journal: Scientific Reports

Article Title: Novel and less invasive biomarker assays to measure liver ATP7B in Wilson disease patients

doi: 10.1038/s41598-025-21455-y

Figure Lengend Snippet: Ratio of ATP7B mRNA levels in Wilson disease (WD) vs. control samples are correlated in fine needle aspiration biopsies (FNA) and core needle biopsies (CNB). Average relative expression was calculated relative to the mean dCt of control samples for the reference gene TF with 3 technical replicates using the comparative Ct method. R 2 is the coefficient of determination (square of the Pearson correlation coefficient r) and p is the P value in the Pearson correlation.

Article Snippet: Anti-ATP7B capture antibody (Abcam, UK, Cat# 131208), anti-ATP7B detection antibody (Novus Biologicals, USA, Cat# NB100-361) and ATP7B recombinant protein (Origene, USA, Cat# TP323635) were purchased from commercial sources.

Techniques: Control, Expressing

Journal: Scientific Reports

Article Title: Novel and less invasive biomarker assays to measure liver ATP7B in Wilson disease patients

doi: 10.1038/s41598-025-21455-y

Figure Lengend Snippet: ATP7B protein is detected by the immunoassay in core needle biopsies (CNBs) but not detectable in fine needle aspiration biopsies (FNAs). Each bar represents the average of 2 technical replicates. Each point represents a single technical replicate.

Article Snippet: Anti-ATP7B capture antibody (Abcam, UK, Cat# 131208), anti-ATP7B detection antibody (Novus Biologicals, USA, Cat# NB100-361) and ATP7B recombinant protein (Origene, USA, Cat# TP323635) were purchased from commercial sources.

Techniques:

Journal: Scientific Reports

Article Title: Therapeutic potential of hepatocyte-like-cells converted from stem cells from human exfoliated deciduous teeth in fulminant Wilson’s disease

doi: 10.1038/s41598-018-38275-y

Figure Lengend Snippet: Hepatic functions and ATP7B expression of SHED-Heps. ( a – e ) In vitro hepatic function assays of SHED-Heps. Culture of SHED-Heps and SHED and measuring of human albumin (hALB), glucose, triglyceride (TG), and urea in the conditioned medium are performed according to the Methods. ( a ) Xenobiotic activity of SHED-Heps and SHED via CYP3A4 is analyzed under dexamethasone stimulation (50 μM). ( b ) Low density lipoprotein (LDL) uptake and bile acid transport are analyzed by DiI-Ac-LDL ( c ) and cholyl-lysyl-fluorescein (CLF) ( d ) staining, respectively. ( e – g ) QRT-PCR shows the expression of ATPase copper transporting beta gene ( ATP7B ) in SHED and SHED-Heps. The results are shown as the ratio to the expression in SHED- Heps ( e ). Immunofluorescent staining demonstrates the expression of ATP7B in SHED and SHED- Heps. Control IgG, isotype-matched IgG staining. ( f ) QRT-PCR shows the effect of ATP7B siRNA treatment in SHED and SHED-Heps. The results are shown as the ratio to the expression of 18S ribosomal RNA ( 18S ). siATP7B, ATP7B siRNA pre-treatment; siCTRL, scrambled control siRNA pre-treatment ( g ) Immunofluorescent staining shows the effect of ATP7B. siRNA treatment in SHED and SHED-Heps. ( h ) ( a , b , e , g ) n = 3 for all groups. **P < 0.01, ***P < 0.005. Graph bars show the means ± SEM. ( c , d , f , h ) Nuclei are stained with DAPI. Bars = 30 μm ( c , d ), 20 μm ( f , h ).

Article Snippet: SHED and SHED-Heps were treated for 48 h with Lipofectamine RNAiMAX (Thermo Fisher Scientific) mixed with human ATP7B siRNA (20 nM; Santa Cruz Biotechnology, Santa Cruz, CA), human stanniocalcin 1 (STC1) siRNA (20 nM; Santa Cruz Biotechnology, Santa Cruz), and the corresponding control scrambled siRNA (Santa Cruz Biotechnology).

Techniques: Expressing, In Vitro, Activity Assay, Staining, Quantitative RT-PCR, Control

Journal: Scientific Reports

Article Title: Therapeutic potential of hepatocyte-like-cells converted from stem cells from human exfoliated deciduous teeth in fulminant Wilson’s disease

doi: 10.1038/s41598-018-38275-y

Figure Lengend Snippet: Suppressive effects and integration of transplanted SHED-Heps on copper accumulated hepatic failure in fulminant LEC rats. SHED and SHED-Heps are transplanted in copper-overloaded LEC rats at 6 weeks of the age. ( a , b ) Histological assays of liver tissues at 10 weeks of the age. Representative images of liver tissues are analyzed by hematoxylin and eosin staining (HE). CV , central vein. ( a ) Copper accumulation is analyzed by Rhodanine staining. Yellow arrows, copper deposition. ( b , c ) Biochemical assay shows the copper contents in the liver tissues at 10 weeks of the age. ( d , e ) Post-transplant kinetics of DiR- labeled SHED and SHED-Hep cells in fulminant LEC rats after 2 weeks (2w) of SHED- and SHED- Hep-transplantation. In vivo tracing shows that DiR labeling is detected in the part of liver of rats. ( d ) Ex vivo tracing shows that DiR labeling is detected in liver and spleen, but not in lung and kidney, of rats. ( e , f , g ) Integration of transplanted SHED- and SHED-Heps in the liver tissues of fulminant LEC rats after 4 weeks of the transplantation. Immunohistochemial assay demonstrates the localization of human albumin (hALB) positive cells in the parenchyma of recipient liver tissues at 10 weeks of the age. Nuclei are stained with hematoxylin. ( f ) Double immunofluorescence shows that localization of human albumin (hALB, red) and human ATP7B (hATP7B, green) in the parenchymal cells of recipient liver tissues of SHED- and SHED-Hep-transplanted fulminant LEC rats. Nuclei are stained with DAPI. ( g ) ( a – g ) LEA, control LEA rats; LEC, non-transplanted fulminant LEC rats; SHED-T, SHED-transplanted fulminant LEC rats; SHED-Hep-T, SHED-Hep-transplanted fulminant LEC rats. ( a , b , f , g ) Bars = 50 μm ( a ), 100 μm ( b , f ), 30 μm ( g ). ( c ) n = 3 for all groups. Graph bars show the means ± SD. *P < 0.05 and ***P < 0.005.

Article Snippet: SHED and SHED-Heps were treated for 48 h with Lipofectamine RNAiMAX (Thermo Fisher Scientific) mixed with human ATP7B siRNA (20 nM; Santa Cruz Biotechnology, Santa Cruz, CA), human stanniocalcin 1 (STC1) siRNA (20 nM; Santa Cruz Biotechnology, Santa Cruz), and the corresponding control scrambled siRNA (Santa Cruz Biotechnology).

Techniques: Staining, Labeling, Transplantation Assay, In Vivo, Ex Vivo, Immunofluorescence, Control

Journal: Scientific Reports

Article Title: Therapeutic potential of hepatocyte-like-cells converted from stem cells from human exfoliated deciduous teeth in fulminant Wilson’s disease

doi: 10.1038/s41598-018-38275-y

Figure Lengend Snippet: In vitro copper metabolism of SHED-Heps. ( a – c ) In vitro copper metabolism assay in SHED-Heps and SHED via ATP7B. SHED, SHED-Heps, and ATP7B-knock-downed SHED-Heps are cultured for 6 h under CuSO 4 (600 μM) treatment and are subsequently cultured without CuSO 4 . Intracellular accumulated copper is measured at indicated time. Biochemical assay shows the intracellular copper contents in SHED and SHED-Heps ( a ), ATP7B- knock-downed SHED ( b ), and ATP7B-knock-downed SHED-Heps ( c ). ( d – f ) I n vitro survival assay in SHED and SHED-Heps via ATP7B under copper stimulation. SHED, SHED-Heps, and ATP7B-knock-downed SHED-Heps are cultured under the different stimulation of CuSO 4 (0, 125, 250, and 500 μM). The cell viability of the cells is measured after 3 days of the stimulation. Biochemical assay shows the viability of SHED and SHED-Heps ( d ), ATP7B-knock-downed SHED ( e ), and ATP7B- knock-downed SHED-Heps ( f ). ( g , h ) ATP7B-siRNA-treated SHED and ATP7B-siRNA-treated SHED-Heps are transplanted into fulminant LEC rats at 6 weeks of the age. The survival of LEC rats transplanted with ATP7B-siRNA-treated SHED (si ATP7B -SHED-T; ( g ), n = 5) and ATP7B-siRNA- treated SHED-Heps (si ATP7B -SHED-Hep-T; ( h ), n = 5) is assay by Kaplan-Meier curve. ( a – f) The results are shown as the ratio to the copper concentration at 0 h in each group. n = 3 for all groups. *P < 0.05 and ***P < 0.005. ns: no significance. Graph bars show the means ± SEM. ( a ) ††† P < 0.005 (vs. SHED at each tipe point). NS: no significance (vs. SHED at each tipe point). ( b , c , e , f ) siATP7B, ATP7B-siRNA treatment; siCTRL, control scrambled siRNA treatment. ( d ) ### P < 0.005 (vs. 0 h of each group). NS: no significance (vs. 0 h of each group). ( g , h ) CTRL-siRNA-SHED-Hep-T, fulminant LEC rats transplanted with control-siRNA-treated SHED (n = 5).

Article Snippet: SHED and SHED-Heps were treated for 48 h with Lipofectamine RNAiMAX (Thermo Fisher Scientific) mixed with human ATP7B siRNA (20 nM; Santa Cruz Biotechnology, Santa Cruz, CA), human stanniocalcin 1 (STC1) siRNA (20 nM; Santa Cruz Biotechnology, Santa Cruz), and the corresponding control scrambled siRNA (Santa Cruz Biotechnology).

Techniques: In Vitro, Cell Culture, Clonogenic Cell Survival Assay, Concentration Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: High Yield Heterologous Expression of Wild-type and Mutant Cu + -ATPase (ATP7B, Wilson Disease Protein) for Functional Characterization of Catalytic Activity and Serine Residues Undergoing Copper-dependent Phosphorylation *

doi: 10.1074/jbc.M109.023341

Figure Lengend Snippet: Construction of the adenovirus vector AdATP7Bmyc: Schematic representation of the linear genome of the rAdATP7Bmyc virus. The shuttle vector containing ATP7B cDNA, preceded by the CMV promoter and including a c- myc tag at the C terminus ( A ), was linearized and subjected to homologous recombination with pAdeasy1 vector ( B ). The dashed line , close to the left end represents the position of the E1, where ATP7Bmyc was inserted. The open box located toward the right end indicates a 2.7-kb deletion in the non-essential E3 region. The shaded boxes at either end represent the left ( L ) and right ( R ) inverted terminal repeats ( ITRs ), and the open box close to the L-ITR represents the encapsidation signal ( ES ). Components of the expression cassette are as follows: P , CMV promoter; ATP7B , ATP7B cDNA; C , c- myc tag; and pA , SV40 polyadenylation signal.

Article Snippet: Recombinant adenovirus vector containing CMV promoter-driven ATP7B cDNA fused with 3′ c- myc tag was constructed as detailed below. pCMV vector containing ATP7B cDNA purchased from Origene was digested with Not1 to release the cDNA and subcloned into pShuttleCMV vector to generate pShuttleCMV-ATP7B. pShuttleCMV-ATP7B plasmid DNA was subjected to PCR amplification at an annealing temperature of 40 °C using the following primers: forward primer, 5′-GGATATTTTGTCCCATTTAT-3′; reverse primer, 5′-ATGCAATCTAGATCACAGGTCCTCCTCTGAGATCAGCTTCTGCTCGATGTACTGCTCCTCATCCC-3′.

Techniques: Plasmid Preparation, Homologous Recombination, Expressing

Journal: The Journal of Biological Chemistry

Article Title: High Yield Heterologous Expression of Wild-type and Mutant Cu + -ATPase (ATP7B, Wilson Disease Protein) for Functional Characterization of Catalytic Activity and Serine Residues Undergoing Copper-dependent Phosphorylation *

doi: 10.1074/jbc.M109.023341

Figure Lengend Snippet: Immunostaining of COS-1 cells infected with rAdATP7Bmyc. Left panel , upper : cells observed 1 day after infection show expressed ATP7B protein adjacent to the nucleus, possibly in the Golgi apparatus. Left panel , lower : cells observed 3 days after infection shows expressed ATP7B protein targeted to intracellular membranes throughout the cytoplasm. Right panel : wide field shows that nearly all cells in culture were infected and express ATP7B, as evidenced by the green color . The cell nuclei of all cells are stained in red . Immunoreaction and staining of rAdATP7Bmyc and nuclei are as described under “Materials and Methods.”

Article Snippet: Recombinant adenovirus vector containing CMV promoter-driven ATP7B cDNA fused with 3′ c- myc tag was constructed as detailed below. pCMV vector containing ATP7B cDNA purchased from Origene was digested with Not1 to release the cDNA and subcloned into pShuttleCMV vector to generate pShuttleCMV-ATP7B. pShuttleCMV-ATP7B plasmid DNA was subjected to PCR amplification at an annealing temperature of 40 °C using the following primers: forward primer, 5′-GGATATTTTGTCCCATTTAT-3′; reverse primer, 5′-ATGCAATCTAGATCACAGGTCCTCCTCTGAGATCAGCTTCTGCTCGATGTACTGCTCCTCATCCC-3′.

Techniques: Immunostaining, Infection, Staining

Journal: The Journal of Biological Chemistry

Article Title: High Yield Heterologous Expression of Wild-type and Mutant Cu + -ATPase (ATP7B, Wilson Disease Protein) for Functional Characterization of Catalytic Activity and Serine Residues Undergoing Copper-dependent Phosphorylation *

doi: 10.1074/jbc.M109.023341

Figure Lengend Snippet: Expression of ATP7B protein in COS-1 cells. Left , electrophoretic (Laemmli) gel with stained protein where lane S shows molecular mass standards (250, 150, 100, 75, 50, and 37 kDa); lanes 1–3 show fractions derived from COS-1 cells infected with adenovirus vector carrying GFP cDNA (sham); lanes 4–6 show fractions derived from COS-1 cells infected with rAdATP7Bmyc. Right : the identity of the expressed ATP7B protein is demonstrated by Western blots using a monoclonal antibody specific for the c- myc tag. Fractions 1 , 4 , and 7 ; 2 , 5 , and 8 ; and 3 , 6 , and 9 were obtained from cell homogenates, by differential centrifugation at increasing speed. Lanes 3 , 6 , and 9 correspond to the microsomal fraction.

Article Snippet: Recombinant adenovirus vector containing CMV promoter-driven ATP7B cDNA fused with 3′ c- myc tag was constructed as detailed below. pCMV vector containing ATP7B cDNA purchased from Origene was digested with Not1 to release the cDNA and subcloned into pShuttleCMV vector to generate pShuttleCMV-ATP7B. pShuttleCMV-ATP7B plasmid DNA was subjected to PCR amplification at an annealing temperature of 40 °C using the following primers: forward primer, 5′-GGATATTTTGTCCCATTTAT-3′; reverse primer, 5′-ATGCAATCTAGATCACAGGTCCTCCTCTGAGATCAGCTTCTGCTCGATGTACTGCTCCTCATCCC-3′.

Techniques: Expressing, Staining, Derivative Assay, Infection, Plasmid Preparation, Western Blot, Centrifugation

Journal: The Journal of Biological Chemistry

Article Title: High Yield Heterologous Expression of Wild-type and Mutant Cu + -ATPase (ATP7B, Wilson Disease Protein) for Functional Characterization of Catalytic Activity and Serine Residues Undergoing Copper-dependent Phosphorylation *

doi: 10.1074/jbc.M109.023341

Figure Lengend Snippet: Electrophoretic analysis of ATP7B and SERCA1 phosphorylation by ATP. Inhibition by BCS and pH stability. ATP7B (50 μg of microsomal protein/ml) was incubated with 50 μ m [γ- 32 P]ATP for 15 min at 37 °C, in a reaction mixture containing 50 m m MES triethanolamine, pH 6.0, 300 m m KCl, 10 m m DTT, 3 m m MgCl 2 , and either 0, 0.5, 1.0, 2.0, or 5 m m BCS. The samples were quenched with 5% trichloroacetic acid, and the sedimented protein was washed with 0.125 n perchloric acid, and solubilized in 2.5% SDS and 0.5% mercaptoethanol, 3% sucrose, and 0.1 mg of bromphenol blue/ml at pH 6.3 or 8.3. A parallel incubation with SERCA1 (30 μg of light sarcoplasmic reticulum vesicles/ml) was performed for 15 s at 3 °C, in a reaction mixture containing 20 m m MOPS, pH 7.0, 80 m m KCl, 3 m m Mg Cl 2 , and 10 μ m CaCl 2 . The solubilized samples (25 μg of protein each) were run on gel electrophoresis with acid ( W–O ) or alkaline (Laemmli) buffer, as described under “Materials and Methods.” The figure shows the radioactive bands and the actual radioactivity counts (●, acid electrophoresis; ■, alkaline electrophoresis) obtained by exposure to a Molecular Dynamics storage phosphor screen.

Article Snippet: Recombinant adenovirus vector containing CMV promoter-driven ATP7B cDNA fused with 3′ c- myc tag was constructed as detailed below. pCMV vector containing ATP7B cDNA purchased from Origene was digested with Not1 to release the cDNA and subcloned into pShuttleCMV vector to generate pShuttleCMV-ATP7B. pShuttleCMV-ATP7B plasmid DNA was subjected to PCR amplification at an annealing temperature of 40 °C using the following primers: forward primer, 5′-GGATATTTTGTCCCATTTAT-3′; reverse primer, 5′-ATGCAATCTAGATCACAGGTCCTCCTCTGAGATCAGCTTCTGCTCGATGTACTGCTCCTCATCCC-3′.

Techniques: Inhibition, Incubation, Nucleic Acid Electrophoresis, Radioactivity, Electrophoresis

Journal: The Journal of Biological Chemistry

Article Title: High Yield Heterologous Expression of Wild-type and Mutant Cu + -ATPase (ATP7B, Wilson Disease Protein) for Functional Characterization of Catalytic Activity and Serine Residues Undergoing Copper-dependent Phosphorylation *

doi: 10.1074/jbc.M109.023341

Figure Lengend Snippet: Protein phosphorylation upon addition of 50 μ m [ γ - 32 P]ATP to ATP7B WT ( A ) and ATP7B mutants ( B ). For the time course of phosphorylation, reaction and quenching at various times, solubilization, and electrophoresis were conducted as explained in the legend for . A , radioactive bands and the actual radioactivity counts obtained by exposure to a Molecular Dynamics storage phosphor screen. B , phosphoenzyme levels obtained following 2-min incubation with 50 μ m [γ- 32 P]ATP, using WT protein, D1027N (putative catalytic phosphorylation site), C983A/C985A (putative transmembrane copper site at TM6), and C575A/c578A (first putative copper site at the NMBD, i.e. closest to the A domain) mutants. Results obtained with acid and alkaline electrophoresis are compared.

Article Snippet: Recombinant adenovirus vector containing CMV promoter-driven ATP7B cDNA fused with 3′ c- myc tag was constructed as detailed below. pCMV vector containing ATP7B cDNA purchased from Origene was digested with Not1 to release the cDNA and subcloned into pShuttleCMV vector to generate pShuttleCMV-ATP7B. pShuttleCMV-ATP7B plasmid DNA was subjected to PCR amplification at an annealing temperature of 40 °C using the following primers: forward primer, 5′-GGATATTTTGTCCCATTTAT-3′; reverse primer, 5′-ATGCAATCTAGATCACAGGTCCTCCTCTGAGATCAGCTTCTGCTCGATGTACTGCTCCTCATCCC-3′.

Techniques: Electrophoresis, Radioactivity, Incubation

Journal: The Journal of Biological Chemistry

Article Title: High Yield Heterologous Expression of Wild-type and Mutant Cu + -ATPase (ATP7B, Wilson Disease Protein) for Functional Characterization of Catalytic Activity and Serine Residues Undergoing Copper-dependent Phosphorylation *

doi: 10.1074/jbc.M109.023341

Figure Lengend Snippet: Decay of [γ- 32 P]phosphoenzyme following a chase with 1 m m non-radioactive ATP in the absence ( A ) or presence ( B ) of 5 m m BCS is shown. WT ATP7B protein was preincubated with 50 μ m [γ- 32 P]ATP for 2 min at 37 °C before addition of 1 m m non-radioactive ATP ( A ), or 1 m m ATP and 5 m m BCS ( B ). In the lower panels , D1027N ( C ) or C983A/C985A ( D ) mutants were preincubated with 50 μ m [γ- 32 P]ATP for 2 min at 37 °C before addition of 1 m m non-radioactive ATP. Samples quenched at serial times were then processed for acid or alkaline electrophoresis as explained for . The values given represent the stoichiometry of phosphorylation based on radioactivity standards determined by scintillation counting and are the difference between results obtained with microsomes derived from COS-1 cells infected with rAdATP7Bmyc and from cells infected with rAdGFP (sham).

Article Snippet: Recombinant adenovirus vector containing CMV promoter-driven ATP7B cDNA fused with 3′ c- myc tag was constructed as detailed below. pCMV vector containing ATP7B cDNA purchased from Origene was digested with Not1 to release the cDNA and subcloned into pShuttleCMV vector to generate pShuttleCMV-ATP7B. pShuttleCMV-ATP7B plasmid DNA was subjected to PCR amplification at an annealing temperature of 40 °C using the following primers: forward primer, 5′-GGATATTTTGTCCCATTTAT-3′; reverse primer, 5′-ATGCAATCTAGATCACAGGTCCTCCTCTGAGATCAGCTTCTGCTCGATGTACTGCTCCTCATCCC-3′.

Techniques: Electrophoresis, Radioactivity, Derivative Assay, Infection

Journal: The Journal of Biological Chemistry

Article Title: High Yield Heterologous Expression of Wild-type and Mutant Cu + -ATPase (ATP7B, Wilson Disease Protein) for Functional Characterization of Catalytic Activity and Serine Residues Undergoing Copper-dependent Phosphorylation *

doi: 10.1074/jbc.M109.023341

Figure Lengend Snippet: Formation of [ γ - 32 P]phosphoenzyme (●) and hydrolytic cleavage of [ γ - 32 P]P i (▴) following addition of 50 μ m [ γ - 32 P]ATP. WT ATP7B (50 μg of microsomal protein/ml) was incubated with 50 μ m [γ- 32 P]ATP at 37 °C, in a reaction mixture containing 50 m m MES triethanolamine, pH 6.0, 300 m m KCl, 10 m m DTT, 3 μ m CuCl 2 , and 3 m m MgCl 2 . Samples were quenched at serial times with 5% trichloroacetic acid and filtered through 0.45-μm Millipore filters under vacuum suction. The filtrate was removed and processed for determination of [ 32 P]P i (see “Materials and Methods”). The filters were then washed three times with 0.125 n cold perchloric acid, and once with cold water, and used for determination of [γ- 32 P]phosphoenzyme (see “Materials and Methods”).

Article Snippet: Recombinant adenovirus vector containing CMV promoter-driven ATP7B cDNA fused with 3′ c- myc tag was constructed as detailed below. pCMV vector containing ATP7B cDNA purchased from Origene was digested with Not1 to release the cDNA and subcloned into pShuttleCMV vector to generate pShuttleCMV-ATP7B. pShuttleCMV-ATP7B plasmid DNA was subjected to PCR amplification at an annealing temperature of 40 °C using the following primers: forward primer, 5′-GGATATTTTGTCCCATTTAT-3′; reverse primer, 5′-ATGCAATCTAGATCACAGGTCCTCCTCTGAGATCAGCTTCTGCTCGATGTACTGCTCCTCATCCC-3′.

Techniques: Incubation

Journal: The Journal of Biological Chemistry

Article Title: High Yield Heterologous Expression of Wild-type and Mutant Cu + -ATPase (ATP7B, Wilson Disease Protein) for Functional Characterization of Catalytic Activity and Serine Residues Undergoing Copper-dependent Phosphorylation *

doi: 10.1074/jbc.M109.023341

Figure Lengend Snippet: Reduction of ex vivo phosphorylated levels by λ -protein phosphatase in vitro , and [ γ - 32 P]phosphoenzyme formation in vitro . WT ATP7B was incubated for 30 min at 30 °C, in the presence (○) or the absence (●) of λ-protein phosphatase (see “Materials and Methods”). Following centrifugation and resuspension in medium 3, ATP7B (50 μg of microsomal protein/ml) was incubated with 50 μ m [γ- 32 P]ATP at 37 °C, in a reaction mixture containing 50 m m MES triethanolamine, pH 6.0, 300 m m KCl, 10 m m DTT, 3 μ m CuCl 2 , and 3 m m MgCl 2 . Samples were quenched at serial times with 5% trichloroacetic acid, and the solubilized protein was subjected to Weber-Osborn electrophoretic analysis. The radioactive bands and the radioactivity counts obtained by exposure to a Molecular Dynamics storage phosphor screen are shown in the figure.

Article Snippet: Recombinant adenovirus vector containing CMV promoter-driven ATP7B cDNA fused with 3′ c- myc tag was constructed as detailed below. pCMV vector containing ATP7B cDNA purchased from Origene was digested with Not1 to release the cDNA and subcloned into pShuttleCMV vector to generate pShuttleCMV-ATP7B. pShuttleCMV-ATP7B plasmid DNA was subjected to PCR amplification at an annealing temperature of 40 °C using the following primers: forward primer, 5′-GGATATTTTGTCCCATTTAT-3′; reverse primer, 5′-ATGCAATCTAGATCACAGGTCCTCCTCTGAGATCAGCTTCTGCTCGATGTACTGCTCCTCATCCC-3′.

Techniques: Ex Vivo, In Vitro, Incubation, Centrifugation, Radioactivity

Journal: The Journal of Biological Chemistry

Article Title: High Yield Heterologous Expression of Wild-type and Mutant Cu + -ATPase (ATP7B, Wilson Disease Protein) for Functional Characterization of Catalytic Activity and Serine Residues Undergoing Copper-dependent Phosphorylation *

doi: 10.1074/jbc.M109.023341

Figure Lengend Snippet: Two-dimensional folding model of the ATP7B sequence, showing serine residues undergoing phosphorylation. The position of phosphorylated serine residues, demonstrated by phosphopeptide analysis using MS, is shown in the ATP7B sequence modeled as proposed by Lutsenko et al. .

Article Snippet: Recombinant adenovirus vector containing CMV promoter-driven ATP7B cDNA fused with 3′ c- myc tag was constructed as detailed below. pCMV vector containing ATP7B cDNA purchased from Origene was digested with Not1 to release the cDNA and subcloned into pShuttleCMV vector to generate pShuttleCMV-ATP7B. pShuttleCMV-ATP7B plasmid DNA was subjected to PCR amplification at an annealing temperature of 40 °C using the following primers: forward primer, 5′-GGATATTTTGTCCCATTTAT-3′; reverse primer, 5′-ATGCAATCTAGATCACAGGTCCTCCTCTGAGATCAGCTTCTGCTCGATGTACTGCTCCTCATCCC-3′.

Techniques: Sequencing