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: Nature Communications

Article Title: Identification of a novel actin-dependent signal transducing module allows for the targeted degradation of GLI1

doi: 10.1038/ncomms9023

Figure Lengend Snippet: ( a ) Autoradiograph of protein microarray showing duplicate spots of recombinant ABLIM1 without (left) or with (right) recombinant DYRK1A, both with radioactive ATP. ( b ) Co-immunoprecipitation of exogenous DYRK1A and ABLIM1 proteins in Hek293T cells. Shown is a representative blot of three independent experiments. ( c ) Cellular actin fractionation assay in NIH3T3 stably expressing empty vector control (mock) or ABLIM1. Jaspla, Jasplakinolide 10 nM for 30 min. Shown is a representative β-actin western blot of two independent experiments (the mean band intensity is given below). ( d ) Cellular actin fractionation assay in NIH3T3 stably expressing empty vector control (mock) or ABLIM2. Jaspla, Jasplakinolide 10 nM for 30 min. Shown is a representative β-actin western blot of two independent experiments (the mean band intensity is given below). ( e ) Deconvolution images of F-actin fibres (phalloidin) in NIH3T3 cells transiently expressing the indicated constructs. Transfected cells were identified through co-transfection of nuclear H2B–GFP. The F-actin promoting Profilin (PFN1) was included as a positive control. Scale bar, 10 μm. ( f ) Quantification of the microscopy experiment depicted in e (mean of two independent experiments with at least 50 cells counted in each experiment). ARR, actin-rich region (lamellipodia, stress fibres, membrane ruffles or pronounced phalloidin positivity). ( g ) Subcellular localization of MKL1-GFP (green) in transiently transfected Hek293A cells. Representative pictures are shown. Nuclei appear in blue (4,6-diamidino-2-phenylindole). Scale bar, 10 μm. ( h ) Quantification of the experiment depicted in f . C, cytoplasmic; N, nuclear; N+C, nuclear and cytoplasmic localization of MKL1-GFP (mean of n =3 independent experiments. At least 100 cells were counted). ( i ) SRE (serum response element) luciferase reporter assay in Hek293T cells transiently transfected with the indicated constructs (mean of n =6±s.d.). ( j ) Phalloidin staining of siRNA-transfected NIH3T3 cells. Shown are representative images of two independent experiments. siABlim1/2: double knockdown of Ablim1 plus Ablim2 . Scale bar, 10 μm. ( k ) Phalloidin staining of siRNA/plasmid double-transfected NIH3T3 cells. The inset in the middle panel should help portrait the different phenotypes observed upon Ablim1 knockdown. siABlim1/2: double knockdown of Ablim1 plus Ablim2. Scale bar, 10 μm. * P <0.05; ** P <0.005; *** P <0.0005 (Student's t -test).

Article Snippet: For the identification of DYRK1A protein substrates, we purchased the ProtoArray Human Protein Microarray v5.0 kinase substrate identification (KSI) kit from Invitrogen (#PAH0525065, containing ∼9,480 recombinant GST-tagged and baculovirus-expressed proteins in duplicate spots).

Techniques: Autoradiography, Microarray, Recombinant, Immunoprecipitation, Fractionation, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Construct, Transfection, Cotransfection, Positive Control, Microscopy, Luciferase, Reporter Assay, Staining

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