Western Blot Protein Detection

Western blotting was performed as previously described (14 (link)) with minor modifications. Cellular proteins were isolated using 1 × RIPA buffer containing a protease inhibitor and a phosphatase inhibitor. The proteins were resolved by electrophoresis in a 10–15% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane (GE Healthcare). The membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween 20. Rabbit monoclonal anti-STING (Cell Signaling Technology, Beverly, MA), Rabbit monoclonal anti-cGAS (Cell Signaling Technology), rabbit polyclonal anti-Rab27b (Bioss Antibodies Inc., Woburn. MA), mouse monoclonal anti-KSHV ORF65 (14 (link)), rabbit polyclonal anti-GAPDH (Cusabio, Houston, TX), rabbit polyclonal anti-calnexin (Bioss Antibodies Inc.), mouse monoclonal anti-HDAC1 (Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-mtTFA (Santa Cruz Biotechnology), rabbit polyclonal anti-MX1 (Bioss Antibodies Inc.), rabbit polyclonal anti-IFIT1 (Bioss Antibodies Inc.), rabbit polyclonal anti-IFIT44L (Bioss Antibodies Inc.) and mouse monoclonal anti-β-actin antibodies (Sigma, St. Louis, MO) were used as primary antibodies. HRP-conjugated anti-rabbit or anti-mouse antibodies (Bethyl Laboratories Inc., Montgomery, TX) were used as secondary antibodies. The results were visualized using an ECL detection reagent (Bio-Rad, Hercules, CA).

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Jeon H., Lee J., Lee S., Kang S.K., Park S.J., Yoo S.M, & Lee M.S. (2019). Extracellular Vesicles From KSHV-Infected Cells Stimulate Antiviral Immune Response Through Mitochondrial DNA. Frontiers in Immunology, 10, 876.

Publication 2019

nitrocellulose membrane Rabbit monoclonal anti-STING Rabbit monoclonal anti-cGAS rabbit polyclonal anti-calnexin mouse monoclonal anti-HDAC1 mouse monoclonal anti-β-actin antibodies HRP-conjugated anti-rabbit or anti-mouse antibodies ECL detection reagent

Actin Anti antibodies Antibodies Buffer Calnexin Cellular Cgas Electrophoresis Gapdh Kshv Membranes Milk Monoclonal antibodies Mouse Mttfa Nitrocellulose Phosphatase Polyacrylamide gel Protease inhibitor Proteins Rabbit Ripa Saline Tween 20

Corresponding Organization : Eulji University

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Variable analysis

dependent variables

Levels of proteins: STING, cGAS, Rab27b, KSHV ORF65, GAPDH, calnexin, HDAC1, mtTFA, MX1, IFIT1, IFIT44L, and β-actin

control variables

Cellular proteins were isolated using 1 × RIPA buffer containing a protease inhibitor and a phosphatase inhibitor
Proteins were resolved by electrophoresis in a 10–15% SDS-polyacrylamide gel
Proteins were transferred onto a nitrocellulose membrane (GE Healthcare)
Membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween 20

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