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