plasmid encoding full-length human ace2  (Addgene inc)

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Sequences of sense strands of double-stranded RNA used to ablate specific protein expression in HEK-ACE2 and Huh7 cells

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Expressing, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Shedding of ACE2 involves loss of its cytoplasmic tail. HEK293 cells were stably transfected with an expression vector encoding full-length ACE2 as described under “Materials and Methods.” OptiMEM containing 0.1 μ m PMA or an equal volume of Me 2 SO carrier was added to exponentially growing cells and collected after 1 h. Following sedimentation of cells, the media were concentrated 10-fold, and 40 μg of media proteins ( M ) were separated by SDS-PAGE (6% v/v) alongside 20 μg of corresponding detergent cell extract ( C ) and immunoblotted with an antibody raised to the ectodomain of ACE2 ( left panel , ectodomain) or the cytosolic tail of ACE2 ( right panel , cytosolic). Immunoreactive bands were visualized with enhanced chemiluminescence as described under “Materials and Methods.”

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Stable Transfection, Transfection, Expressing, Plasmid Preparation, Sedimentation, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Shed ACE2 occurs as two distinct glycoforms. HEK-ACE2 cells were incubated in OptiMEM containing 0.1 μ m PMA or an equal volume of Me 2 SO carrier for 1 h, and the media were collected and concentrated as described. Media proteins ( M ; 40 μg) or detergent cell extracts ( C ; 20 μg) were incubated at 37 °C for 16 h in the presence or absence of endoglycosidase H ( Endo H ) or PNGase F and subsequently separated by SDS-PAGE. Following electrotransfer, immunoblotting was carried out using an antibody to the ectodomain of ACE2 as described under “Materials and Methods.”

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Incubation, SDS Page, Electrotransfer, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Shedding of ACE2 is inhibited by broad spectrum hydroxamate-based metalloprotease inhibitors. HEK-ACE2 cells were incubated in OptiMEM containing various concentrations of the secretase inhibitors TAPI-1 or GM6001 or an equal volume of carrier (Me 2 SO). After 15 min, PMA (0.1 μ m ) or an equal volume of Me 2 SO was added, and incubation was continued for 1 h. The medium was subsequently harvested and concentrated as described; cells were pelleted and detergent extracts were collected as described under “Materials and Methods.” A , media proteins ( upper panel , 40 μg) and cell lysates ( lower panel , 20 μg) were separated by SDS-PAGE and immunoblotted for ACE2. Immunoreactive bands were visualized by enhanced chemiluminescence. B , graphical representation of results of densitometric analysis of three such experiments, ± S.E. Black shading , –PMA; gray shading , +PMA.

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Incubation, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: PMA-stimulated ACE2 shedding is sensitive to ADAM17 inhibition. HEK-ACE2 cells were incubated for 15 min in the presence of the ADAM10 inhibitor GI254023X or the mixed ADAM10/ADAM17 inhibitor GW280264X or an equal volume of Me 2 SO. Subsequently, incubation was continued in the presence of PMA (0.1 μ m ). Media were harvested and concentrated as described under “Materials and Methods.” A , media proteins (40 μg) were separated by SDS-PAGE and immunoblotted for ACE2. B , graphical representation of results of densitometric analysis of three such experiments, ± S.E.

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Inhibition, Incubation, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Stimulated ACE2 shedding is inhibited by NTIMP3 but not by NTIMP1. HEK-ACE2 cells were incubated for 15 min in the presence of NTIMP1, NTIMP3, or an equal volume of phosphate-buffered saline. Subsequently, incubation was continued in the presence of PMA (0.1 μ m ). Media were harvested and concentrated as described. A , media proteins (40 μg) were separated by SDS-PAGE and immunoblotted for ACE2. B , graphical representation of results of densitometric analysis of the immunoblots of three such experiments, ± S.E.

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Incubation, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Ablation of ADAM17 expression by siRNA reduces stimulated ACE2 shedding. HEK-ACE2 cells were transiently transfected with a mixture of double-stranded RNA derived from the coding sequence of ADAM10, ADAM17, or the control sequence GL2 as described under “Materials and Methods.” Twenty-four hours after transfection, cells were incubated in OptiMEM containing 0.1 μ m PMA for 1 h. Media were concentrated as described, and cell lysates were prepared. A , media proteins (40 μg) and cell lysates (50 μg) were separated by SDS-PAGE and immunoblotted for ACE2, ADAM10, and ADAM17, as indicated. Mock , mock transfection. B , graphical representation of densitometric analysis of immunoblots of media ACE2 from three such experiments, ± S.E.

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Expressing, Transfection, Derivative Assay, Sequencing, Incubation, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Overexpression of ADAM17 increases PMA-stimulated ACE2 shedding. HEK-ACE2 cells were transiently transfected with an expression vector encoding ADAM9, ADAM10, or ADAM17, as described under “Materials and Methods.” Thirty-six hours after transfection, cells were incubated in OptiMEM containing 0.1 μ m PMA for 1 h. Media were concentrated as described, and detergent cell extracts were harvested. A , media proteins (40 μg) and detergent cell extracts (50 μg) were separated by SDS-PAGE and immunoblotted for ACE2 ( upper panel , media), ADAM9, ADAM10, or ADAM17 ( lower panels , lysates). Membranes were stripped and reprobed for β-actin as a loading control. B , graphical representation of densitometric analysis of immunoblots of media ACE2 from three such experiments, ± S.E.

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Incubation, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: PMA-stimulated endogenous ACE2 shedding is sensitive to ADAM17 inhibition. A , OptiMEM containing 0.1 μ m PMA or an equal volume of Me 2 SO carrier was added to exponentially growing Huh7 cells and collected after 6 h. Following sedimentation of cells, the media were concentrated, and 100 μg of media proteins ( M ) was separated by SDS-PAGE (4–12% v/v) alongside 50 μg of corresponding detergent cell extract ( C ) and immunoblotted with an antibody raised to the ectodomain of ACE2 ( left panel , ectodomain) or the cytosolic tail of ACE2 ( right panel , cytosolic). Immunoreactive bands were visualized with enhanced chemiluminescence as described under “Materials and Methods.” B , Huh7 cells were incubated for 15 min in the presence of 50 μ m GM6001 or 1 μ m the ADAM10 inhibitor GI254023X or the mixed ADAM10/ADAM17 inhibitor GW280264X or an equal volume of Me 2 SO. Subsequently, incubation was continued for 4 h in the presence or absence of PMA (0.1 μ m ). Media were harvested and concentrated as described under “Materials and Methods.” Concentrated media samples (20 μg) were assayed for their ability to cleave an ACE2-specific fluorogenic substrate, as described under “Materials and Methods.” Black shading , –PMA; gray shading , +PMA.

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Inhibition, Sedimentation, SDS Page, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Ablation of ADAM17 expression by siRNA reduces regulated endogenous ACE2 shedding. Huh7 cells (50% confluent) were transfected with double-stranded RNA oligomers (as described under “Materials and Methods”) to ADAM10, ADAM17, or a negative control sequence. After 40 h, the media were aspirated and replaced with OptiMEM containing 100 n m PMA or an equal volume of Me 2 SO. After a further 5-h incubation, the media were concentrated, and cell lysates were prepared as described under “Materials and Methods.” Cell lysates (50 μg) were separated by SDS-PAGE and immunoblotted for ADAM10 ( A ) and ADAM17 ( B ). Concentrated media samples (20 μg) were assayed for their ability to cleave an ACE2-specific fluorogenic substrate as described under “Materials and Methods.”

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Expressing, Transfection, Negative Control, Sequencing, Incubation, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Tumor Necrosis Factor-α Convertase (ADAM17) Mediates Regulated Ectodomain Shedding of the Severe-acute Respiratory Syndrome-Coronavirus (SARS-CoV) Receptor, Angiotensin-converting Enzyme-2 (ACE2) *

doi: 10.1074/jbc.M505111200

Figure Lengend Snippet: Overexpression of ADAM17 increases PMA-stimulated endogenous ACE2 shedding. HEK-ACE2 cells were transiently transfected with an expression vector encoding ADAM9, ADAM10, or ADAM17, as described under “Materials and Methods.” Thirty-six hours after transfection, cells were incubated in OptiMEM containing 0.1 μ m PMA for 4 h. Media were concentrated as described, and detergent cell extracts were harvested. A , detergent cell extracts (50 μg) were separated by SDS-PAGE and immunoblotted for ADAM9, ADAM10, or ADAM17. Membranes were stripped and reprobed for β-actin as a loading control. B , concentrated media samples (20 μg) were assayed for their ability to cleave an ACE2-specific fluorogenic substrate as described under “Materials and Methods.”

Article Snippet: Plasmid Construction— cDNA encoding full-length human ACE2 (GenBank™ accession number AB046569) was amplified from a human kidney cDNA library (Clontech) using the primer pair 5′-ACGTCCATGTCAAGCTCTTCCTGGCTCCTTCTC-3′ (forward) and 5′-CTAGGCTAAAAGGAGGTCTGAACATCATCAGTGTTT-3′ (reverse), digested, and ligated into the XbaI and XhoI sites of the pCI-Neo expression vector (Promega).

Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Incubation, SDS Page