Journal: PLoS Pathogens

Article Title: Lu/BCAM Adhesion Glycoprotein Is a Receptor for Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1)

doi: 10.1371/journal.ppat.1003884

Figure Lengend Snippet: GST-CNF1 was incubated with buffer or with recombinant BCAM in a molar ratio CNF1∶rBCAM of 1∶1, 1∶10 and 1∶100, respectively for 20 min. The mixture was added to HeLa cells. Following 2 h incubation the cells were lysed and the CNF1-catalysed deamidation of RhoA was analyzed by the shift of the modified GTPase in SDS-PAGE by Western-blotting (A). HeLa cells were incubated with an anti Lu/BCAM antibody (AB B12) that binds to the extracellular domain or as control with an anti-Lu/BCAM antibody (AB C16) directed against the intracellular part of the glycoprotein. GST-CNF1 was then added to the cells for 2 h. We followed the toxins uptake by the amount of modified RhoA (shift in SDS-PAGE, B). Shown is a typical result of 3 independent experiments. Colocalization of DyLight488-labeled GST-CNF1 with Lu/BCAM and EEA1 (C) Top: HeLa-cells were treated on ice with DyLight488-labeled GST-CNF1 (5 mg/ml) (green) for 30 min to allow receptor binding. After 30 min cells were transferred to 37° for 30 min to induce uptake. Subsequently cells were fixed and stained for EEA1 (red) by immunofluorescence. Images are from the middle of the confocal stack. The image on the right is a maximal projection of all confocal planes. Scale bar indicates 10 µm. Middle: HeLa-cells were treated with labeled GST-CNF1 as in A. After fixation cells were stained for Lu/BCAM (red) by immunofluorescence. Images are from the middle of the confocal stack. The image on the right is a magnification of the white box. Scale bar indicates 10 µm. Bottom: HeLa-cells were treated as in A, but instead of GST-CNF1, cells were treated with DyLight488-labeled GST-CNFY (5 mg/ml) (green). After fixation cells were stained for Lu/BCAM (red) by immunofluorescence. Images are from the middle of the confocal stack. The image on the right is a magnification of the white box. Scale bar indicates 10 µm. Shown is a typical staining of 9 HeLa cells analyzed.

Article Snippet: For these studies we used recombinant Lu/BCAM, which contains a C-terminal human IgG domain for purification (Sino biology).

Techniques: Incubation, Recombinant, Modification, SDS Page, Western Blot, Labeling, Binding Assay, Staining, Immunofluorescence

Journal: PLoS Pathogens

Article Title: Lu/BCAM Adhesion Glycoprotein Is a Receptor for Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1)

doi: 10.1371/journal.ppat.1003884

Figure Lengend Snippet: A) K562 leukemia cells, which do not express Lu/BCAM (top) and the isogenic cell line K562-Lu/BCAM expressing the receptor (bottom), were treated with GST-CNF1 for different time periods from 1 h to overnight (ON) as indicated. Uptake of the toxin was analyzed by the shift of modified RhoA in SDS-PAGE. B) GST-CNF1 was incubated with buffer or with recombinant BCAM in a molar ratio CNF1∶rBCAM of 1∶1, 1∶10 and 1∶100, respectively for 20 min. The mixture was added to K562-Lu/BCAM cells for 2 h. Cells were lysed and the deamidation of RhoA was analyzed by the shift of the modified GTPase in SDS-PAGE by Western-blotting. Data are representative for at least 3 independent experiments.

Article Snippet: For these studies we used recombinant Lu/BCAM, which contains a C-terminal human IgG domain for purification (Sino biology).

Techniques: Expressing, Modification, SDS Page, Incubation, Recombinant, Western Blot

Journal: PLoS Pathogens

Article Title: Lu/BCAM Adhesion Glycoprotein Is a Receptor for Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1)

doi: 10.1371/journal.ppat.1003884

Figure Lengend Snippet: A) For dot-blots 5 µl of 3 µM solutions of GST-CNFs, GST-CNF fragments and GST alone, respectively, were spotted onto a nitrocellulose membrane. The membrane was blocked with skimmed milk and recombinant BCAM (6 µM) was added for 1 h at room temperature. Following washing bound rBCAM was detected with an anti-Lu/BCAM antibody. Equal protein load was analyzed by visualizing the GST part of the spotted proteins with an anti GST-antibody. B) Biacore protein-protein interaction studies: An antibody against human IgG (Millipore) was coupled to two lanes of a CM5-biacore chip. As ligand recombinant BCAM containing a C-terminal human IgG domain (Sino biologics) was exclusively guided over lane 2. In a second step, GST-CNF proteins as analyte were guided over both lanes. Bound protein is given as relative units (RU) corrected for the unspecific binding to lane 1 as average plus standard deviation of three independent experiments.

Article Snippet: For these studies we used recombinant Lu/BCAM, which contains a C-terminal human IgG domain for purification (Sino biology).

Techniques: Recombinant, Binding Assay, Standard Deviation

Journal: Nucleic Acids Research

Article Title: The N-terminus of the human RecQL4 helicase is a homeodomain-like DNA interaction motif

doi: 10.1093/nar/gks591

Figure Lengend Snippet: The Sld2-homologous N-terminal domain of RecQL4 interacts directly with TopBP1. ( A ) Schematic depiction of human RecQL4 and TopBP1. ( B ) Specific interaction of the N-terminal domain of RecQL4 fused to the GAL4 DNA-BD with C-terminal part (a.a. 793–1522) of TopBP1 fused to the GAL4-AD in yeast. Interaction was semi-quantitatively assessed by β-galactosidase liquid culture activity assays. β-galactosidase values represent the mean and standard deviations of three independent experiments. ( C ) GST pull-down experiments performed with purified GST, GST–TopBP1 and GST–TopBP1 (a.a. 793–1522) and [ 35 S]–methionine–labelled RecQL4 (a.a. 1–675) (lower panel). Coomassie brilliant blue R250-stained SDS–PAGE gels of the purified GST constructs are presented (upper panel). ( D ) GST pull-down experiments performed with purified GST and GST–RecQL4 (a.a. 1–54) and [ 35 S]-cysteine-labelled full-length TopBP1. Coomassie brilliant blue R250-stained SDS–PAGE gels of the purified GST constructs are presented (upper panel). Input IVT: 10 µl of the in vitro translation product. ( E ) Reciprocal immunoprecipitation of RecQL4 and TopBP1. Cell extracts from human Hek293 cells transiently co-transfected with expression vectors for RecQL4 and ToPBP1 were subjected to immunoprecipitation using antibodies against RecQL4 and TopBP1 as described in ‘Material and Methods’. The precipitates were then analysed by SDS–PAGE and Western blot against the cognate proteins. Controls without antibody or with non-specific rabbit IgG served as negative control and 10% of the input served as an indicator for the (co-)immunoprecipitation efficiency.

Article Snippet: TopBP1 polyclonal antibody (4 μg, Novus Biologicals), and anti-RecQL4 (a rabbit polyclonal antibody raised against recombinant a.a. 1–115 of human RecQL4) or pre-immune serum were incubated with 20 μl (bed volume) of protein G-Sepharose (GE Healthcare) and 1% BSA in lysis buffer overnight at 4°C.

Techniques: Activity Assay, Purification, Staining, SDS Page, Construct, In Vitro, Immunoprecipitation, Transfection, Expressing, Western Blot, Negative Control

Journal: Nucleic Acids Research

Article Title: The N-terminus of the human RecQL4 helicase is a homeodomain-like DNA interaction motif

doi: 10.1093/nar/gks591

Figure Lengend Snippet: Characterization of the TopBP1 interaction with RecQL4. ( A ) Identification of the RecQL4_N54 BD of TopBP1 by GST pull-down. Pull-down experiments were performed as in C–D with GST-fusion constructs as bait for in vitro translated proteins as indicated. Only TopBP1 (a.a. 1233–1522) shows efficient binding to GST-RecQL4_N54. Purified GST and GST-TopBP1 (a.a. 1233–1264) stained with Coomassie brilliant blue R250 are shown on the right. ( B ) Real-time in vitro SPR binding analysis of RecQL4_N54 to anti-GST antibody captured GST-TopBP1 (a.a. 1233–1522). Sensorgrams of 200, 100, 50, 25, 12.5, 6.25 and 3.13 µM RecQL4_N54 binding injected in triplicate (black lines) are shown overlaid with the best fit derived from a 1:1 interaction model (red lines) (left). Fit of the equilibrium data for TopBP1 (a.a. 1233–1522) binding (middle). The purified GST-TopBP1 (a.a. 1233–1593) used for the experiment stained with Coomassie brilliant blue R250 is shown on the right.

Article Snippet: TopBP1 polyclonal antibody (4 μg, Novus Biologicals), and anti-RecQL4 (a rabbit polyclonal antibody raised against recombinant a.a. 1–115 of human RecQL4) or pre-immune serum were incubated with 20 μl (bed volume) of protein G-Sepharose (GE Healthcare) and 1% BSA in lysis buffer overnight at 4°C.

Techniques: Construct, In Vitro, Binding Assay, Purification, Staining, Injection, Derivative Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nox2 and Nox4 mediate tumour necrosis factor-α-induced ventricular remodelling in mice

doi: 10.1111/j.1582-4934.2011.01261.x

Figure Lengend Snippet: Gene symbol, assay ID and RefSeq for RT-PCR

Article Snippet: Mouse Nox2, Nox4 and ANP proteins were examined using purified anti-gp91phox (BD Biosciences, San Diego, CA, USA), anti-Nox4 antibody (Novus Biologicals, Littleton, CO, USA) and anti-ANP (Santa Cruz Biotechnology Inc.), following the procedures previously described [ ].

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nox2 and Nox4 mediate tumour necrosis factor-α-induced ventricular remodelling in mice

doi: 10.1111/j.1582-4934.2011.01261.x

Figure Lengend Snippet: Upregulation of Nox2 and Nox4 mRNA and protein in ventricles of TNF-α-injected mice. Total RNA was extracted from the ventricular tissues of TNF-α-injected and control mice and Nox2 and Nox4 mRNA levels were examined using real-time PCR. Nox2 (A) and Nox4 mRNA levels (B) were shown. Nox2 and Nox4 protein levels were determined by immunoblotting. A representative of Nox2 and Nox4 proteins were shown (C). Densitometric analysis of Nox2 (D) and Nox4 protein (E) normalized with GAPDH were shown. C, control; T, TNF-α-injected mice. * P < 0.05.

Article Snippet: Mouse Nox2, Nox4 and ANP proteins were examined using purified anti-gp91phox (BD Biosciences, San Diego, CA, USA), anti-Nox4 antibody (Novus Biologicals, Littleton, CO, USA) and anti-ANP (Santa Cruz Biotechnology Inc.), following the procedures previously described [ ].

Techniques: Injection, Control, Real-time Polymerase Chain Reaction, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nox2 and Nox4 mediate tumour necrosis factor-α-induced ventricular remodelling in mice

doi: 10.1111/j.1582-4934.2011.01261.x

Figure Lengend Snippet: Nox2 and Nox4 mediate TNF-α-induced upregulation of IL-1β and IL-6 genes and ROS production. Human adult cardiomyocytes were transfected with Nox2 and Nox4 siRNA using siPORT Amine transfection reagents. Total RNA was extracted from human adult cardiomyocytes 24 hrs after transfection. Human Nox2 and Nox4 mRNA levels were examined using real-time PCR. Nox2 (A) and Nox4 (B) mRNA levels after siRNA treatment were shown. In the other experiments, cells were treated with TNF-α (20 ng/ml) for 6 hrs at 37°C after treatment with Nox2 and Nox4 siRNA. IL-1β (C) and IL-6 mRNA (D) levels were examined with real-time PCR. TNF-α-induced ROS production in human adult cardiomyocytes was determined with CM-H 2 DCFDA following Nox2 and Nox4 siRNA treatment (E). Data were expressed as relative light unit (RLU) ± S.D. * P < 0.05 versus control values. # P < 0.05 versus TNF-α treatment values.

Article Snippet: Mouse Nox2, Nox4 and ANP proteins were examined using purified anti-gp91phox (BD Biosciences, San Diego, CA, USA), anti-Nox4 antibody (Novus Biologicals, Littleton, CO, USA) and anti-ANP (Santa Cruz Biotechnology Inc.), following the procedures previously described [ ].

Techniques: Transfection, Real-time Polymerase Chain Reaction, Control

Journal: PLoS ONE

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue

doi: 10.1371/journal.pone.0163710

Figure Lengend Snippet: (A) An autoubiquitination assay was performed using UCP at various time points. GST-UCP (0.2 μg) was incubated at 37°C in the presence of E1 and Flag-ubiquitin, and autoubiquitination was visualized with anti-Flag antibody. (B) The catalytic ability of UncH5c proteins was analyzed in an in vitro autoubiquitination assay. GST-UbcH5c proteins (0.2 μg) and GST-UCP (0.2 μg) were incubated at 37°C for 40 min in the presence of E1 and Flag-ubiquitin, and the ubiquitinated proteins were detected by immunoblotting using anti-Flag antibody. (C) The lysine-specific linkage of UCP was defined using lysine-to-arginine ubiquitin mutants (K6R, K11R, K48R and K63R), single-lysine ubiquitin mutants (K6, K11, K48 and K63) and a lysine-null ubiquitin mutant (K-null). Autoubiquitination assays were performed using GST-UCP (0.2 μg) and wild type ubiquitin or ubiquitin mutants at 37°C for 1 h, and ubiquitinated proteins were detected by immunoblotting using anti-ubiquitin antibody. (D) GST-UCP (0.2 μg) and His-UCP C95A (2 μg) were incubated at 37°C for 1 h in the presence of E1 and Flag-ubiquitin. His-UCP C95A was then pulled down with Ni-NTA agarose, and His-UCP C95A polyubiquitination was detected by immunoblotting using anti-Flag antibody.

Article Snippet: The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

Techniques: Incubation, In Vitro, Western Blot, Mutagenesis

Journal: PLoS ONE

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue

doi: 10.1371/journal.pone.0163710

Figure Lengend Snippet: (A) His-UCP WT (1 μg) and GST-UCP WT (1 μg) or truncated UCP mutants (UCP N-term , UCP Core , UCP C-term , UCP ∆N and UCP ∆C ) (1 μg) were incubated at 4°C for 2 h. His-UCP was then pulled down with Ni-NTA agarose at 4°C for 2 h, and the bound domains were detected by immunoblotting. (B) His-VHL (1 μg) and GST-UCP WT (1 μg) or truncated UCP mutants (1 μg) were incubated at 4°C for 2 h. His-UCP was then pulled down with Ni-NTA agarose, and the bound domains were detected by immunoblotting. (C) HA-VHL (5 μg) and GST-UCP WT (5 μg) or truncated UCP mutant plasmids (5 μg, each) were co-transfected into HEK-293T cells. GST-UCP proteins were pulled down with GST-resin, and then HA-VHL was detected by immunoblotting. (D) The plasmids Flag-UCP WT (5 μg) and GST-VHL WT or truncated VHL mutants (β, α, ∆ECEB) (5 μg, each) were co-transfected into HEK-293T cells. GST-VHL was pulled down with GST-agarose, and then interaction with UCP was detected by immunoblotting using anti-Flag antibody. (E, F) Schematic representation of UCP and pVHL. The functional domains of UCP and pVHL are delineated by three colored boxes (white, gray and black). The ability of the different domains to bind UCP and VHL is indicated; +; binding; -; no binding.

Article Snippet: The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

Techniques: Incubation, Western Blot, Mutagenesis, Transfection, Functional Assay, Binding Assay

Journal: PLoS ONE

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue

doi: 10.1371/journal.pone.0163710

Figure Lengend Snippet: (A) The catalytic activity of UCP WT (0.2 μg) and truncated UCP mutants (UCP N-term , UCP Core , UCP C-term , UCP ∆N and UCP ∆C ) (0.2 μg) was assessed using an in vitro ubiquitination assay. The proteins were incubated at 37°C for 1 h in the presence of E1 and His-ubiquitin, and the ubiquitinated proteins were detected by immunoblotting using anti-His antibody. (B) The substrate region of UCP for autoubiquitination was analyzed in stable HeLa cell lines transfected with shRNA-control or shRNA-UCP. Different stable HeLa cell lines were co-transfected with plasmids encoding each truncated UCP mutant (5 μg) and HA-Ubiquitin (2 μg), treated with 10μM MG132 for 12 h and harvested at 48 h post-transfection. The cells were then lysed, and UCP was pulled down with GST-agarose. The ubiquitinated domains were subsequently detected by immunoblotting using anti-HA antibody. (C) GST-tagged UCP WT (0.2 μg) and truncated UCP mutants (UCP C-term and UCP ∆C , 0.2 μg) were mixed with His-VHL (2 μg) and subjected to an in vitro ubiquitination assays at 37°C for 1 h. pVHL was pulled down with Ni-NTA agarose, and ubiquitinated forms were detected by immunoblotting using anti-Flag antibody. (D) HA-VHL (5 μg) and GST-wild-type UCP or truncated UCP mutant plasmids (5 μg) were co-transfected into HEK-293T cells and treated with/without 10 μM MG132 for 12 h. Changes in the pVHL expression level were then detected by immunoblotting.

Article Snippet: The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

Techniques: Activity Assay, In Vitro, Ubiquitin Assay, Incubation, Western Blot, Transfection, shRNA, Mutagenesis, Expressing

Journal: PLoS ONE

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue

doi: 10.1371/journal.pone.0163710

Figure Lengend Snippet: (A) In vitro ubiquitination assays were performed using His-UCP WT as the enzyme and inactive UCP mutants containing lysine-to-arginine mutations as the substrates (GST-UCP-C95A, GST-UCP- C95A/K76R, GST-UCP-C95A/K100R, and GST-UCP-C95A/K76R,K100R) (2 μg). After the reaction, GST-UCP was pulled down with GST agarose at 4°C for 2 h. The ubiquitinated forms were visualized with anti-Flag antibody. (B) An in vitro ubiquitination assay was performed using His-UCP WT (0.2 μg) and wild-type and/or single-lysine pVHL mutants (VHL K159 , VHL K171 , VHL K196 and VHL ∆K ) (2 μg) at 37°C for 1 h. After incubation, pVHL was pulled down with GST agarose, and ubiquitinated forms were detected by immunoblotting using anti-Flag antibody. (C) Wild-type pVHL (5 μg), single-lysine pVHL (5 μg) mutant and HA-Ubiquitin plasmids (2 μg) were co-transfected into HEK-293T cells, which were then incubated with 10 μM MG132 for 12 h. After the cells were lysed, pVHL was pulled down with GST agarose, and ubiquitinated forms were detected by immunoblotting with anti-HA antibody.

Article Snippet: The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

Techniques: In Vitro, Ubiquitin Assay, Incubation, Western Blot, Mutagenesis, Transfection

Journal: PLoS ONE

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue

doi: 10.1371/journal.pone.0163710

Figure Lengend Snippet: (A) An autoubiquitination assay was performed using His-UCP WT and His-UCP C118A (0.2 μg each) along with wild-type ubiquitin (Ub WT ) at various time points. Ubiquitinated forms were detected by immunoblotting using anti-ubiquitin antibody. (B) An autoubiquitination assay was performed using His-UCP WT and His-UCP C118A (0.2 μg each) along with lysine-null ubiquitin (Ub ∆K ) at various time points. Ubiquitinated forms were detected by immunoblotting using anti-ubiquitin antibody. (C) In vitro ubiquitination assay was performed using His-UCP WT (0.2 μg) and GST-UCP C95A (2 μg) at various time points. GST-UCP C95A was pulled down using GST agarose and then polyubiquitination was separated by SDS-PAGE under denaturing or non-denaturing (without β-mercaptoethanol) condition. The polyubiquitin chains were detected by anti-Flag antibody. (D) Illustration of the expected reaction steps for polyubiquitination by UCP in the trans manner and the status of polyubiquitin chains on the substrate under different conditions. (E) An in vitro ubiquitination assay was performed using wild-type UCP (His-UCP WT ), double-mutant UCP with K78R and K100R (His-UCP K76R/K100R ), UCP-C118A mutant (His-UCP C118A ) (0.2 μg each) and GST-UCP C95A (2 μg) at 37°C for 1 h in reaction buffer. After the reaction, GST-UCP C95A was pulled down with GST agarose, and polyubiquitination was analyzed by immunoblotting under denaturing (+β-mercaptoethanol) or non-denaturing (-β-mercaptoethanol) conditions using anti-Flag antibody.

Article Snippet: The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

Techniques: Western Blot, In Vitro, Ubiquitin Assay, SDS Page, Mutagenesis

Journal: PLoS ONE

Article Title: E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue

doi: 10.1371/journal.pone.0163710

Figure Lengend Snippet: (A) Autoubiquitination was assessed using GST-UCP C95A (1 μg) and GST-UCP ∆N (0.5, 1, or 2 μg), and polyubiquitination was analyzed by immunoblotting using anti-Flag antibody. (B) An in vitro ubiquitination assay was performed using the protein pair GST-UCP ∆N /GST-UCP C95A (each 0.2 μg) and His-VHL (2 μg). His-VHL protein was pulled down with Ni-NTA agarose, and ubiquitinated forms were analyzed by immunoblotting using anti-Flag antibody. (C) HA-VHL and UCP WT or UCP mutants (UCP C95A , UCP C118A and UCP ∆N ) or various pairs of UCP mutant (UCP C95A /UCP ΔN , UCP ΔN /UCP C118A ) plasmids (total of 10 μg) were co-transfected into HEK-293T cells and treated with/without 10 μM MG132 for 12 h. At 48 h post-transfection, the cells were harvested and lysed. Changes in pVHL expression levels were detected by immunoblotting. (D) An in vitro ubiquitination assay was performed using wild-type UCP (0.2 μg) or UCP mutants (GST-UCP C118A and GST-UCP ∆N , each 0.2 μg) and His-UCP C95A (2 μg). His-UCP C95A protein was pulled down with Ni-NTA agarose, and ubiquitinated forms of His-UCP C95A were assessed by immunoblotting using anti-Flag antibody. (E) An in vitro ubiquitination assay was performed using wild-type UCP (0.2 μg), double-mutant UCP with K76R and K100R (His-UCP K76R/K100R, 0.2 μg), C118A-mutant UCP (His-UCP C118A, 0.2 μg) and GST-UCP C95A or GST-UCP CA (2 μg) at 37°C for 1 h. The GST-UCP C95A or GST-UCP CA protein was pulled down with GST agarose and analyzed by immunoblotting using anti-Flag antibody.

Article Snippet: The GST-tagged proteins were then purified from the cleared lysates by affinity purification using Glutathione Sepharose beads (GE Healthcare Life Sciences).

Techniques: Western Blot, In Vitro, Ubiquitin Assay, Mutagenesis, Transfection, Expressing

Journal: Cell Reports

Article Title: AMPK regulates Bcl2-L-13-mediated mitophagy induction for cardioprotection

doi: 10.1016/j.celrep.2024.115001

Figure Lengend Snippet:

Article Snippet: Haspin Recombinant Protein , SignalChem , Cat# G10-11G-05.

Techniques: Transduction, Virus, Recombinant, Western Blot, Protease Inhibitor, Immunoprecipitation, Purification, ATP Assay, In Situ, Kinase Assay, Real-time Polymerase Chain Reaction, Knock-Out, Knock-In, Negative Control, Software

Journal: Frontiers in Microbiology

Article Title: Production of SARS-CoV-2 N Protein-Specific Monoclonal Antibody and Its Application in an ELISA-Based Detection System and Targeting the Interaction Between the Spike C-Terminal Domain and N Protein

doi: 10.3389/fmicb.2021.726231

Figure Lengend Snippet: Production and characterization of the anti-SARS-CoV-2 N protein-specific monoclonal antibody. (A) Expression of biotin peptide-6 × His-tagged SARS-CoV-2 N protein (SARS-CoV-2 N-Bio-His 6 ). Recombinant SARS-CoV-2 N-Bio-His 6 protein was expressed in ExpiCHO cells and purified from cell culture supernatants using Ni-NTA agarose chromatography. The purified recombinant protein was analyzed by SDS-PAGE (left) and western blotting with peroxidase-conjugated streptavidin (middle) and anti-His-tag antibody (right). Arrowhead, biotin peptide-6 × His-tagged SARS-CoV-2 N protein; R, reducing condition; NR, non-reducing condition. (B) Expression of biotin peptide-6 × His-tagged MERS-CoV N protein (MERS-CoV N-Bio-His 6 ). The purified recombinant protein was analyzed by SDS-PAGE. Arrowhead, biotin peptide-6 × His-tagged MERS-CoV N protein. (C) The recombinant SARS-CoV-2 N-Bio-His 6 protein and CpG-DNA were combined in a DOPE:CHEMS complex and the complex was injected intraperitoneally into BALB/c mice ( n = 4) three times. ELISA was performed with mouse sera to determine whether recombinant SARS-CoV-2 N-Bio-His 6 protein-specific antibody was present. (D) Ascites were collected from mice injected with cloned hybridoma cells (1G10C4). ELISA was performed with the ascites to determine whether recombinant SARS-CoV-2 N-Bio-His 6 protein-specific antibody was present. (E) The monoclonal antibody was purified from the ascitic fluid using Protein-A column chromatography and analyzed using SDS-PAGE. HC, heavy chain; LC, light chain. (F) Subclasses of the monoclonal antibody were identified by ELISA. (G) The detection limit of the monoclonal antibody against SARS-CoV-2 N-Bio-His 6 protein was measured by ELISA. (H) Binding of the monoclonal antibody to recombinant SARS-CoV-2 N-Bio-His 6 protein was measured by ELISA.

Article Snippet: For the immunoprecipitation assay, SARS-CoV- 2-, MERS- CoV-, or HCoV-OC43-infected Vero cell lysates were incubated with the SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb) at 4 ° C for 2 h. Immunocomplexes were isolated with Protein A beads (Repligen, Waltham, MA, United States) and analyzed by western blotting with rabbit anti-SARS-CoV-2 N protein polyclonal antibody (Catalog No. 40588-T62; Sino Biological, Vienna, Austria).

Techniques: Expressing, Recombinant, Purification, Cell Culture, Chromatography, SDS Page, Western Blot, Injection, Enzyme-linked Immunosorbent Assay, Clone Assay, Column Chromatography, Binding Assay

Journal: Frontiers in Microbiology

Article Title: Production of SARS-CoV-2 N Protein-Specific Monoclonal Antibody and Its Application in an ELISA-Based Detection System and Targeting the Interaction Between the Spike C-Terminal Domain and N Protein

doi: 10.3389/fmicb.2021.726231

Figure Lengend Snippet: Specificity of the anti-SARS-CoV-2 N protein monoclonal antibody. (A) Analysis of monoclonal antibody specificity. Recombinant SARS-CoV-2 N-Bio-His 6 protein or recombinant MERS-CoV N-Bio-His 6 protein was captured on streptavidin-coated 96-well immunoplates and then incubated with anti-SARS-CoV-2 N protein monoclonal antibody. Reactivity of the monoclonal antibody to each recombinant protein was determined by ELISA. (B) MERS- CoV-, SARS-CoV- 2-, or HCoV-OC43-infected and non-infected Vero cell lysates were immunoblotted with the anti-SARS-CoV-2 N protein monoclonal antibody (clone 1G10C4 mAb). β-Actin was used as the loading control. (C) MERS- CoV-, SARS-CoV- 2-, or HCoV-OC43-infected and non-infected Vero cell lysates were immunoprecipitated with normal mouse IgG or the anti-SARS-CoV-2 N protein monoclonal antibody (clone 1G10C4 mAb). The immunocomplexes were subjected to western blot analysis using rabbit anti-SARS-CoV-2 N protein antibody (Catalog No. 40588-T62; Sino Biological).

Article Snippet: For the immunoprecipitation assay, SARS-CoV- 2-, MERS- CoV-, or HCoV-OC43-infected Vero cell lysates were incubated with the SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb) at 4 ° C for 2 h. Immunocomplexes were isolated with Protein A beads (Repligen, Waltham, MA, United States) and analyzed by western blotting with rabbit anti-SARS-CoV-2 N protein polyclonal antibody (Catalog No. 40588-T62; Sino Biological, Vienna, Austria).

Techniques: Recombinant, Incubation, Enzyme-linked Immunosorbent Assay, Infection, Immunoprecipitation, Western Blot

Journal: Frontiers in Microbiology

Article Title: Production of SARS-CoV-2 N Protein-Specific Monoclonal Antibody and Its Application in an ELISA-Based Detection System and Targeting the Interaction Between the Spike C-Terminal Domain and N Protein

doi: 10.3389/fmicb.2021.726231

Figure Lengend Snippet: Detection of N protein in cells infected with SARS-CoV-2 S, GH, or GR clade with anti-SARS-CoV-2 N protein-specific monoclonal antibody. (A,B) Western blot analyses for the detection of N protein in cells infected with SARS-CoV-2 S, GH, or GR clade. Vero cells (A) and Calu-3 cells (B) were infected with SARS-CoV-2 S, GH, or GR clade at a MOI of 0.1 for 72 h. Cell lysates were analyzed by western blotting with the anti-SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb). β-Actin was used as the loading control. (C) Confocal microscopy was used to detect N protein in cells infected with SARS-CoV-2 S, GH, or GR clade. Vero cells were cultured on cover glass in 12-well plates and infected with SARS-CoV-2 S, GH, or GR clade at a MOI of 0.1 for 48 h. The infected Vero cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The cells were incubated with anti-SARS-CoV-2 N protein-specific monoclonal antibody or normal IgG and then with Alexa Fluor 488–conjugated goat anti-mouse IgG. Nuclei were stained with Hoechst 33258. Images were obtained by confocal microscope. Scale bar, 10 μm.

Article Snippet: For the immunoprecipitation assay, SARS-CoV- 2-, MERS- CoV-, or HCoV-OC43-infected Vero cell lysates were incubated with the SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb) at 4 ° C for 2 h. Immunocomplexes were isolated with Protein A beads (Repligen, Waltham, MA, United States) and analyzed by western blotting with rabbit anti-SARS-CoV-2 N protein polyclonal antibody (Catalog No. 40588-T62; Sino Biological, Vienna, Austria).

Techniques: Infection, Western Blot, Confocal Microscopy, Cell Culture, Incubation, Staining, Microscopy

Journal: Frontiers in Microbiology

Article Title: Production of SARS-CoV-2 N Protein-Specific Monoclonal Antibody and Its Application in an ELISA-Based Detection System and Targeting the Interaction Between the Spike C-Terminal Domain and N Protein

doi: 10.3389/fmicb.2021.726231

Figure Lengend Snippet: Detection of N protein in SARS-CoV-2 S, GH, or GR clade virus particles with anti-SARS-CoV-2 N protein-specific monoclonal antibody. (A) Western blot analyses. Vero cells were infected with SARS-CoV-2 S, GH, or GR clade at a MOI of 0.01 for 72 h and then cell culture supernatants were collected. The cell culture supernatants were lysed with cell lysis buffer and analyzed by western blotting with the anti-SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb). Virus titers were measured by plaque assay. (B) Immunoprecipitation analysis. Vero cells were infected with SARS-CoV-2 S, GH, or GR clade at a MOI of 0.01 for 72 h and then cell culture supernatants were collected. The cell culture supernatants were lysed with cell lysis buffer and immunoprecipitated with normal mouse IgG or the anti-SARS-CoV-2 N protein monoclonal antibody (clone 1G10C4 mAb). The immunocomplexes were subjected to western blot analysis using rabbit anti-SARS-CoV-2 N protein antibody (Catalog No. 40588-T62; Sino Biological).

Article Snippet: For the immunoprecipitation assay, SARS-CoV- 2-, MERS- CoV-, or HCoV-OC43-infected Vero cell lysates were incubated with the SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb) at 4 ° C for 2 h. Immunocomplexes were isolated with Protein A beads (Repligen, Waltham, MA, United States) and analyzed by western blotting with rabbit anti-SARS-CoV-2 N protein polyclonal antibody (Catalog No. 40588-T62; Sino Biological, Vienna, Austria).

Techniques: Western Blot, Infection, Cell Culture, Lysis, Plaque Assay, Immunoprecipitation

Journal: Frontiers in Microbiology

Article Title: Production of SARS-CoV-2 N Protein-Specific Monoclonal Antibody and Its Application in an ELISA-Based Detection System and Targeting the Interaction Between the Spike C-Terminal Domain and N Protein

doi: 10.3389/fmicb.2021.726231

Figure Lengend Snippet: N protein in SARS-CoV-2 S, GH, or GR clade virus particles interact with SARS-CoV-2 Spike CD. (A) Expression of SARS-CoV-2 Spike CD-Fc. The recombinant Fc control protein and the SARS-CoV-2 Spike CD-Fc fusion protein were expressed in ExpiCHO cells, purified from cell culture supernatants using Protein A column chromatography and analyzed by SDS-PAGE and Coomassie blue staining. (B) Interaction of the N protein in virus particles with SARS-CoV-2 Spike CD. Vero cells were infected with SARS-CoV-2 S, GH, or GR clade at a MOI of 0.01 for 72 h and then cell culture supernatants were collected. Cell culture supernatants were lysed with cell lysis buffer and incubated with Fc or SARS-CoV-2 Spike CD-Fc. Fc-bound proteins were pulled down with Protein A beads and subjected to western blot analysis using the anti-SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb). Western blot of virus lysates is shown as a control.

Article Snippet: For the immunoprecipitation assay, SARS-CoV- 2-, MERS- CoV-, or HCoV-OC43-infected Vero cell lysates were incubated with the SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb) at 4 ° C for 2 h. Immunocomplexes were isolated with Protein A beads (Repligen, Waltham, MA, United States) and analyzed by western blotting with rabbit anti-SARS-CoV-2 N protein polyclonal antibody (Catalog No. 40588-T62; Sino Biological, Vienna, Austria).

Techniques: Expressing, Recombinant, Purification, Cell Culture, Column Chromatography, SDS Page, Staining, Infection, Lysis, Incubation, Western Blot

Journal: Frontiers in Microbiology

Article Title: Production of SARS-CoV-2 N Protein-Specific Monoclonal Antibody and Its Application in an ELISA-Based Detection System and Targeting the Interaction Between the Spike C-Terminal Domain and N Protein

doi: 10.3389/fmicb.2021.726231

Figure Lengend Snippet: Interaction between SARS-CoV-2 Spike CD-Fc and SARS-CoV-2 N-Bio-His 6 recombinant proteins. (A) Schematic of the bait and prey assay system. (B) Immunoplates (96-well) were coated with streptavidin and then recombinant SARS-CoV-2 N-Bio-His 6 protein was added to each well. After addition of MERS-CoV Spike CD-Fc or SARS-CoV-2 Spike CD-Fc, the amount of CoV Spike CD-human Fc fusion protein bound to recombinant SARS-CoV-2 N-Bio-His 6 protein in the wells was determined by ELISA. (C) Specificity of the interaction between SARS-CoV-2 Spike CD-Fc and SARS-CoV-2 N-Bio-His 6 recombinant protein. Serially diluted non-biotinylated-recombinant SARS-CoV-2 N-His 6 protein was incubated with SARS-CoV-2 Spike CD-Fc and then added to the wells containing SARS-CoV-2 N-Bio-His 6 -coated streptavidin. The extent of the competition was measured by ELISA using HRP-conjugated anti-human IgG Fc antibody.

Article Snippet: For the immunoprecipitation assay, SARS-CoV- 2-, MERS- CoV-, or HCoV-OC43-infected Vero cell lysates were incubated with the SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb) at 4 ° C for 2 h. Immunocomplexes were isolated with Protein A beads (Repligen, Waltham, MA, United States) and analyzed by western blotting with rabbit anti-SARS-CoV-2 N protein polyclonal antibody (Catalog No. 40588-T62; Sino Biological, Vienna, Austria).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Frontiers in Microbiology

Article Title: Production of SARS-CoV-2 N Protein-Specific Monoclonal Antibody and Its Application in an ELISA-Based Detection System and Targeting the Interaction Between the Spike C-Terminal Domain and N Protein

doi: 10.3389/fmicb.2021.726231

Figure Lengend Snippet: Detection of SARS-CoV-2 in cell culture media using SARS-CoV-2 N protein-specific monoclonal antibody and recombinant SARS-CoV-2 Spike CD-Fc protein. (A) Schematic of the ELISA. (B) SARS-CoV-2 clade S and HCoV-OC43 and (C) SARS-CoV-2 clade GH and clade GR in cell culture supernatants were lysed with cell lysis buffer and serially diluted in PBST. These virus lysates were added into 96-well immunoplates coated with SARS-CoV-2 N protein-specific monoclonal antibody. After incubation for 2 h at room temperature, recombinant SARS-CoV-2 Spike CD-Fc protein was added, and then HRP-conjugated anti-human IgG Fc antibody was added to each well. The amount of SARS-CoV-2 N protein in each well was determined by ELISA.

Article Snippet: For the immunoprecipitation assay, SARS-CoV- 2-, MERS- CoV-, or HCoV-OC43-infected Vero cell lysates were incubated with the SARS-CoV-2 N protein-specific monoclonal antibody (clone 1G10C4 mAb) at 4 ° C for 2 h. Immunocomplexes were isolated with Protein A beads (Repligen, Waltham, MA, United States) and analyzed by western blotting with rabbit anti-SARS-CoV-2 N protein polyclonal antibody (Catalog No. 40588-T62; Sino Biological, Vienna, Austria).

Techniques: Cell Culture, Recombinant, Enzyme-linked Immunosorbent Assay, Lysis, Incubation