Journal: Bioengineering

Article Title: COVID-19 Diagnostic Strategies Part II: Protein-Based Technologies

doi: 10.3390/bioengineering8050054

Figure Lengend Snippet: The developed protein microarray-based tests for COVID-19 detection.

Article Snippet: PEPperPRINT GmbH [ ] , PEPperCHIP ® Pan-Corona Spike Protein Microarray , Antibodies against S antigen , S proteins derived from seven coronaviruses translated into overlapping peptides , (No info) , (No info) , One array with 4564 peptides in duplicate , RUO , For Serum antibody fingerprint analysis, Immune monitoring and Epitope studies.

Techniques: Microarray, Peptide Microarray, Bioprocessing, Derivative Assay, High Throughput Screening Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Virus, Clinical Proteomics

Journal: Molecular and Cellular Biology

Article Title: Spatial Regulation of Cyclic AMP-Epac1 Signaling in Cell Adhesion by ERM Proteins

doi: 10.1128/mcb.00463-10

Figure Lengend Snippet: FIG. 2. Epac1 directly interacts with ERM proteins. (A) HA-Epac1 coimmunoprecipitation (IP) with Flag-ezrin, Flag-radixin, and Flag-moesin in HEK293 cells. TL, total lysate. (B) Coimmunoprecipitation of Flag-radixin with YFP-tagged wild-type Epac1, Epac11-49, and Epac1DEP in HEK293 cells. The 49 N-terminal amino acids of Epac1 are required for the interaction with radixin. (C) Coimmunoprecipitation of Flag-radixin with HA-tagged versions of Epac1 or Epac2 in HEK293 cells. In contrast to HA-Epac1, HA-Epac2 is unable to coimmunoprecipitate Flag-radixin.

Article Snippet: The mouse monoclonal green fluorescent protein (GFP) antibody was obtained from Roche, Flag M2 antibody was from SigmaAldrich, monoclonal V5 antibody was from Invitrogen, phospho-ERM (ezrin T567, radixin T564, moesin T558) antibody was from Cell Signaling Technology, monoclonal HA antibody was from Covance (HA11), and monoclonal radixin antibody was from BD Biosciences.

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Spatial Regulation of Cyclic AMP-Epac1 Signaling in Cell Adhesion by ERM Proteins

doi: 10.1128/mcb.00463-10

Figure Lengend Snippet: FIG. 3. ERM proteins require the open conformation to bind Epac1. (A) Domain architecture of radixin. The FERM (4.1 protein, ezrin, radixin, moesin) domain and actin binding domain (ABD) form an intramolecular interaction and are linked by an -helical region. Phosphor- ylation of T564 is required for the open conformation of radixin, and I577 and I580 are part of the hydrophobic interaction surface between the ABD and the FERM domain. (B) Coimmunoprecipitation of HA-Epac1 with Flag-tagged wild-type radixin and radixin lacking the ABD (Flag-radixinABD, residues 1 to 492) in HEK293 cells. The separate N terminus of radixin shows enhanced binding to Epac1 compared to full-length radixin, implying that the presence of the C-terminal ABD suppresses binding of radixin to Epac1. (C) Coimmunoprecipitation of HA-Epac1 with wild-type Flag-radixin and the constitutively open mutants Flag-radixin(T564D) and Flag-radixin(I577D, F580D) in HEK293 cells. The amount of coimmunoprecipitated HA-Epac1 is significantly increased with these radixin mutants. (D) Live imaging of YFP-tagged wild-type radixin and the constitutively open mutant radixin(T564D) in HEK293 cells, showing that upon conformational opening radixin is redistributed to the PM. (E) Live imaging of Epac1 in HEK293 cells. The indicated constructs were expressed alone or with Flag-radixin(T5645D. (F) 007-AM (1 M) treatment of HEK293 cells transfected with the FRET sensor CFP-Epac1DEP-YFP. The sensor reports the cAMP-induced conforma- tional change as a loss of intramolecular FRET; deletion of the DEP domain precludes changes in intermolecular FRET due to cAMP-dependent relocalization (51). When this Epac1 FRET construct is recruited to the PM by coexpressed radixin(T564D), its conformational opening upon 007-AM treatment remains unaffected. Scale bars (all images), 10 m.

Article Snippet: The mouse monoclonal green fluorescent protein (GFP) antibody was obtained from Roche, Flag M2 antibody was from SigmaAldrich, monoclonal V5 antibody was from Invitrogen, phospho-ERM (ezrin T567, radixin T564, moesin T558) antibody was from Cell Signaling Technology, monoclonal HA antibody was from Covance (HA11), and monoclonal radixin antibody was from BD Biosciences.

Techniques: Binding Assay, Imaging, Mutagenesis, Construct, Transfection

Journal: Molecular and Cellular Biology

Article Title: Spatial Regulation of Cyclic AMP-Epac1 Signaling in Cell Adhesion by ERM Proteins

doi: 10.1128/mcb.00463-10

Figure Lengend Snippet: FIG. 4. GPCR-mediated activation of ERM proteins induces binding of Epac1. (A) Coimmunoprecipitation of HA-Epac1 with Flag-radixin in HEK293 cells stimulated with thrombin (0.2 U/ml, 2 min). Thrombin stimulation results in increased phosphorylation of the ERM proteins (ezrin T567, radixin T564, and moesin T558) (right) and the enhanced interaction of HA-Epac1 with Flag-radixin. (B) Quantification of the relative binding of Epac1 to radixin from three independent experiments, as performed for panel A. Statistical analysis was performed using a one-tailed Student t test. (C) Measurement of FRET between YFP-radixin and Epac1-TdTom or Epac11-49-TdTom in HEK293 cells during stimulation with thrombin receptor-activating peptide (TRP; 50 M). FRET between YFP-radixin and Epac1-TdTom, expressed as ratio of TdTom/YFP fluorescence, increases upon TRP addition, confirming that binding to Epac1 is increased when radixin is driven in the open conformation. The traces are representative of three independent measurements. The average increase (standard deviation) in the TdTom/YFP ratio was 6.0% 1.1%. (D) Coimmunoprecipitation of endogenous ezrin with endogenous Epac1 in ACHN cells with or without prestimulation with sphingosine 1-phosphate (S1P; 1 M) to induce the conformational opening of ezrin.

Article Snippet: The mouse monoclonal green fluorescent protein (GFP) antibody was obtained from Roche, Flag M2 antibody was from SigmaAldrich, monoclonal V5 antibody was from Invitrogen, phospho-ERM (ezrin T567, radixin T564, moesin T558) antibody was from Cell Signaling Technology, monoclonal HA antibody was from Covance (HA11), and monoclonal radixin antibody was from BD Biosciences.

Techniques: Activation Assay, Binding Assay, Phospho-proteomics, One-tailed Test, Standard Deviation

Journal: Molecular and Cellular Biology

Article Title: Spatial Regulation of Cyclic AMP-Epac1 Signaling in Cell Adhesion by ERM Proteins

doi: 10.1128/mcb.00463-10

Figure Lengend Snippet: FIG. 5. GPCR-mediated activation of ERM proteins induces a clustered localization of Epac1 at the PM. (A) Live imaging of Epac1-YFP before and after 50 M TRP stimulation to induce phosphorylation and conformational opening of ERM proteins in HEK293 cells. In response to TRP, Epac1-YFP has a clustered localization at the PM. Quantitative analysis reveals the rapid kinetics of TRP-induced recruitment of Epac1 in this experiment. Shown are fluorescence intensities at the right flank of the plasma membrane (PM; red) and in the cytosol (blue) as well as the PM/cytosol ratio (green). (B) Live imaging of Epac11-49-YFP before and after 50 M TRP stimulation in HEK293 cells. Epac11-49-YFP is not targeted to the PM in response to TRP. (C) Live imaging of YFP-radixin and Epac1-TdTom after 50 M TRP stimulation to induce phosphorylation and conformational opening of radixin in HEK293 cells. In response to TRP, both constructs are similarly clustered at the PM. (D) Subcellular localization of Epac1-YFP and endogenous radixin after 50 M TRP stimulation in HEK293 cells. (E) Live imaging of HEK293 cells that were transfected with CFP-N49 and Epac11-49-YFP and stimulated with TRP (50 M) and 007-AM (1 M). Both constructs accumulate at the PM but show different subcellular distributions. (F) Live imaging of Epac1-YFP in HEK293 cells, showing the inhibition of plasma membrane recruitment by TRP (50 M) by preincubation with the exoenzyme C3, an inhibitor of RhoA (16 h, 30 g/ml). (G) Live imaging of Epac1-YFP in HEK293 cells, showing the recruitment of Epac1 to the plasma membrane by coexpression of the catalytic domain of p190-RhoGEF. The clustered localization of Epac1 remains upon activation of Epac1 by stimulation with 007-AM (1 M). Scale bars (all images), 10 m.

Article Snippet: The mouse monoclonal green fluorescent protein (GFP) antibody was obtained from Roche, Flag M2 antibody was from SigmaAldrich, monoclonal V5 antibody was from Invitrogen, phospho-ERM (ezrin T567, radixin T564, moesin T558) antibody was from Cell Signaling Technology, monoclonal HA antibody was from Covance (HA11), and monoclonal radixin antibody was from BD Biosciences.

Techniques: Activation Assay, Imaging, Phospho-proteomics, Clinical Proteomics, Membrane, Construct, Transfection, Inhibition

Journal: Molecular and Cellular Biology

Article Title: Spatial Regulation of Cyclic AMP-Epac1 Signaling in Cell Adhesion by ERM Proteins

doi: 10.1128/mcb.00463-10

Figure Lengend Snippet: FIG. 6. ERM binding is required for efficient Epac1-mediated cell adhesion. (A) Coimmunoprecipitation of HA-Epac1 with Flag-radixin in the presence of the V5-tagged C-terminal ABD of radixin in HEK293 cells. Overexpression of V5-ABD decreases the interaction of HA-Epac1 with Flag-radixin. (B) Live imaging of YFP-Epac1 in HEK293 cells coexpressed with CFP-radixin ABD. In the presence of CFP-radixin ABD, plasma membrane recruitment by TRP (50 M) is inhibited, whereas the subsequent 007-AM (1 M)-induced translocation of Epac1 remained unaffected. (C) Adhesion of Jurkat T cells that stably express Epac1 transiently transfected with either empty vector (EV) or the ABD of radixin to compete for binding of Epac1 to endogenous ERM proteins. Transfected cells were allowed to adhere to a fibronectin-coated surface for 45 min, and adhesion was subsequently detected by measuring the activity of cotransfected luciferase. Adhesion induced by activation of Epac1 with 007 (100 M) is decreased by overexpression of the ABD of radixin. Shown are average data with standard deviations from three individual experiments, with 007-induced adhesion in EV-transfected cells normalized to 100%. Statistical analysis was performed using a one-tailed Student t test. The Western blot labeled with the V5 and Epac1 antibody (5D3) shows the expression of Epac1 and the transfected V5-radixin ABD construct. (D) Adhesion of Jurkat T cells transiently transfected with YFP-1-49-Epac1 and either EV or the ABD of radixin. Adhesion of transfected cells was measured by a method similar to the method used for Fig. 5C. Results show that the radixin ABD does not reduce adhesion induced by YFP-1-49-Epac1. Shown are average data with standard deviations from three individual experiments, with 007-induced adhesion in EV-transfected cells normalized to 100%. Statistical analysis was performed using a one-tailed Student t test. The Western blot labeled with the V5 and Epac1 antibody (5D3) shows the expression of YFP-1-49-Epac1 and the V5-radixin ABD construct. (E) Adhesion of Ovcar3 cells transfected with either control or ezrin, radixin, and moesin SMARTpool siRNAs. Sixty hours after siRNA transfection, cells were allowed to adhere to a fibronectin-coated surface for 45 min in the absence or presence of 100 M 007, and adhesion was subsequently detected by measuring endogenous phosphatase activity. Shown are average data with standard deviations from two individual experiments, normalized to adhesion of control siRNA-transfected cells. Statistical analysis was performed using a one-tailed Student t test. The Western blot shows the expression of endogenous ezrin, radixin, moesin, and tubulin (as a loading control).

Article Snippet: The mouse monoclonal green fluorescent protein (GFP) antibody was obtained from Roche, Flag M2 antibody was from SigmaAldrich, monoclonal V5 antibody was from Invitrogen, phospho-ERM (ezrin T567, radixin T564, moesin T558) antibody was from Cell Signaling Technology, monoclonal HA antibody was from Covance (HA11), and monoclonal radixin antibody was from BD Biosciences.

Techniques: Binding Assay, Over Expression, Imaging, Clinical Proteomics, Membrane, Translocation Assay, Stable Transfection, Transfection, Plasmid Preparation, Activity Assay, Luciferase, Activation Assay, One-tailed Test, Western Blot, Labeling, Expressing, Construct, Control