protoarray human protein microarray v5 0  (Thermo Fisher)

Journal: Molecules and Cells

Article Title: Hepatitis C Virus Nonstructural 5A Protein Interacts with Telomere Length Regulation Protein: Implications for Telomere Shortening in Patients Infected with HCV

doi: 10.14348/molcells.2021.0167

Figure Lengend Snippet: (A) Identification of TEN1 as a binding partner for NS5A in protein microarray. (B) HEK293T cells were cotransfected with Myc-tagged NS5A and Flag-tagged TEN1 expression plasmid. At 48 h after transfection, total cell lysates were immunoprecipitated (IP) with an anti-Myc monoclonal antibody, and then bound protein was detected by immunoblot analysis with an anti-Flag monoclonal antibody. Arrowhead indicates IgG heavy chain. (C) Schematic illustration of both wild-type and mutants of HCV NS5A expression plasmid. WT, wild type; aa, amino acids. (D) HEK293T cells were cotransfected with Flag-tagged TEN1 and Myc-tagged NS5A expression plasmids. At 48 h after transfection, cell lysates were immunoprecipitated with an anti-Flag polyclonal antibody, and bound proteins were immunoblotted with an anti-Myc polyclonal antibody. Protein expressions of Myc-tagged NS5A and Flag-tagged TEN1 were verified by immunoblotting with an anti-Myc or anti-Flag monoclonal antibody using the same cell lysates.

Article Snippet: Firstly, ProtoArray ® Human Protein Microarray v5.0 (Invitrogen) was incubated with blocking buffer (50 mM HEPES [pH 7.5], 25% glycerol, 0.08% Triton X-100, 200 mM NaCl, 20 mM reduced glutathione, and 0.1 mM dithiothreitol [DTT]) for 1 h at 4°C.

Techniques: Binding Assay, Microarray, Expressing, Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Identification of Novel Focal Adhesion Kinase Substrates: Role for FAK in NFκB Signaling

doi: 10.7150/ijbs.10273

Figure Lengend Snippet: Microarray screen for FAK substrates. a. Left: Autoradiograph image of control and FAK protein microarray slides. Right: Control microarray containing 1, 5, 10 and 25 ng/dot of GST or GST-Paxillin. b. Spot-pair signals from control and FAK slides block #40 (upper panels), with identified proteins, microarray positions and relative signal intensities (average pixels/pair) below. A-F, proteins with increased phosphorylation by FAK relative to control; G, positive control protein, PKCeta; H-J , proteins with equal or decreased phosphorylation levels in the FAK slide compared to control.

Article Snippet: The following reagents/kits were used: ProtoArray Human Protein Microarray v5.0 Kinase Substrate Identification Complete Kit (Invitrogen), full-length active Src (SignalChem), GST-FAK (Invitrogen), baculovirus-encoded Pyk2 (gift of Vita Golubovskaya, Roswell Park Cancer Institute [RPCI]), purified vitronectin (Advanced BioMatrix), purified GST-PTPN5[a.a.17-565] (ProteinTech), FAK inhibitor PF-573,228 (Sigma), TNFα (Abcam), Recombinant Light (Enzo Life Sciences).

Techniques: Microarray, Autoradiography, Blocking Assay, Positive Control

Journal: Journal of Neuroinflammation

Article Title: A new Purkinje cell antibody (anti-Ca) associated with subacute cerebellar ataxia: immunological characterization

doi: 10.1186/1742-2094-7-21

Figure Lengend Snippet: Probing of a commercial protein microarray revealed strong binding of the patient's sera to human ARHGAP26 (Panel A) . In accordance with this finding, binding of IgG from the patient's serum (Lane P), but not from three healthy controls (Lane C1-C3), to a recombinant human full length ARHGAP26 protein was found (Panel B). Western blotting confirmed the presence of ARHGAP26 in primate cerebellar extract (Panel C, lane 2), and incubation with the patient's CSF resulted in a band running at the same height as the ARHGAP26 band (Panel C, lane 1); the significance of the additional band recognized by the commercial antibody is unknown. The commercial ARHGAP26 antibody bound to the Purkinje cell layer and the molecular layer (Panel D) and showed a good overlay with the patient's IgG depicted in yellow (Panel E). Finally, preadsorption of the patient's CSF with the ARHGAP26 protein (Panel H), but not preadsorption with a control protein (Panel G), resulted in complete loss of binding to cerebellar tissue sections in an indirect immunofluorescence assay; panel F shows binding of IgG from a non-preadsorbed aliquot of the same CSF sample (exposure time was 2 sec in all cases to detect also low fluorescence signals).

Article Snippet: A commercially available human protein microarray (Protoarray v5.0; Invitrogen) spotted with >9000 human full length-proteins purified from a baculovirus-based expression system was probed with the patient's serum according to the manufacturer's instructions.

Techniques: Microarray, Binding Assay, Recombinant, Western Blot, Incubation, Immunofluorescence, Fluorescence

Journal: The EMBO Journal

Article Title: SCF Fbxw5 targets kinesin‐13 proteins to facilitate ciliogenesis

doi: 10.15252/embj.2021107735

Figure Lengend Snippet: Coomassie‐stained SDS–polyacrylamide gel electrophoresis (PAGE) of different proteins used in the assay. Cul1 was obtained using a split and co‐express system (in which the C‐ and N‐terminal domains are co‐expressed as individual proteins and therefore run as two distinct bands at around 50 kDa) followed by in vitro neddylation. SCF Fbxw5 complexes were prepared by mixing equimolar amounts of Fbxw5/Skp1 and Cul1˜Nedd8/Rbx1 sub‐complexes. Numbers left of the gel indicate molecular weight marker in kilo‐Dalton (kDa, same accounts for all following gels and blots). Workflow and example of a sub‐array of the protoarray screen. Protein microarrays containing more than 9,000 human proteins spotted in duplicates were incubated with 15 µM FITC‐labelled ubiquitin (Ub), 100 nM E1 (Uba1‐His 6 ), E2s (0.5 µM each of UbcH5b and Cdc34) and 0.15 µM SCF Fbxw5 for 1.5 h at 37°C. Right panel shows overlay of a selected sub‐array probed with (red) or without (green) SCF Fbxw5 complexes. White box marks the established substrate Sas6. Comparison of protoarray signal intensities of candidate substrates probed with or without SCF Fbxw5 complexes. Sas6, Sec23b and MCAK are marked as red dots (other published substrates (e.g. Ask1, Eps8) were not among the 9,000 proteins spotted on the array). Note that axes have different scaling. Cellular components GO analysis of identified substrates using DAVID webtool with protoarray proteins as background. Validation of individual targets by manually curated in vitro ubiquitylation experiments. HA‐tagged (hemagglutinin) candidate proteins were purified from Hek293T cells via anti‐HA immunoprecipitation (IP) followed by HA‐peptide elution. Candidates were incubated with 20 µM His 6 ‐Ubiquitin, 170 nM E1, E2s (0.5 µM each of UbcH5b and Cdc34) and 5 mM ATP in the presence or absence of 0.1 µM SCF Fbxw5 for 2 h at 37°C. Substrates were detected via SDS–PAGE followed by Western blotting using anti‐HA antibodies for detection. Data information: Source data are presented in Dataset . Source data are available online for this figure.

Article Snippet: In order to identify novel substrates of the SCF Fbxw5 complex, we performed an in vitro ubiquitylation screen on commercial protein microarrays (ProtoArray ® v5.0, Thermo Fisher Scientific) containing more than 9,000 human proteins expressed and purified as GST‐fusion proteins from insect cells.

Techniques: Staining, Polyacrylamide Gel Electrophoresis, In Vitro, Molecular Weight, Marker, Incubation, Ubiquitin Proteomics, Comparison, Biomarker Discovery, Purification, Immunoprecipitation, SDS Page, Western Blot

Journal: The EMBO Journal

Article Title: SCF Fbxw5 targets kinesin‐13 proteins to facilitate ciliogenesis

doi: 10.15252/embj.2021107735

Figure Lengend Snippet: Scatter plots comparing intensities of duplicate SCF Fbxw5 (left) and control (right) samples. Dotted lines indicate the cut‐off for candidate substrates (500 AU). Comparison of all protoarray signal intensities probed with or without SCF Fbxw5 complexes. Co‐IP of endogenous HGS, Sec23b, CSRP2 and GSTP1 with exogenously expressed Flag‐Fbxw5 using anti‐Flag antibodies. Co‐IP of endogenous HGS and Sec23b with endogenous Fbxw5 as in (C), except that anti‐Fbxw5 antibodies were used instead of anti‐Flag. Data information: Source data are presented in Dataset . Source data are available online for this figure.

Article Snippet: In order to identify novel substrates of the SCF Fbxw5 complex, we performed an in vitro ubiquitylation screen on commercial protein microarrays (ProtoArray ® v5.0, Thermo Fisher Scientific) containing more than 9,000 human proteins expressed and purified as GST‐fusion proteins from insect cells.

Techniques: Control, Comparison, Co-Immunoprecipitation Assay