Western Blot Analysis of Cellular Proteins

Total protein was isolated from cells using RIPA lysis buffer (Thermo Fisher Scientific) and Halt protease inhibitor cocktail (Thermo Fisher Scientific). Protein concentration was measured using the Pierce BCA protein assay kit (Thermo Fisher Scientific) and cell lysates containing 20 µg of total protein were loaded and separated in 4-20% TGX precast SDS-PAGE gels (Bio-Rad, Hercules, CA) followed by transfer onto PVDF membranes (Bio-Rad). Immunoblot analyses were carried out as previously described (19 (link)). Mouse monoclonal antibody against GFP (1:1000, Santa Cruz, Dallas, Texas) and HRP-conjugated mouse monoclonal antibody against β-actin (1:50,000, Sigma-Aldrich, St. Louis, MO) were used for the assay.

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Geng H., Subramanian S., Wu L., Bu H.F., Wang X., Du C., De Plaen I.G, & Tan X.D. (2021). SARS-CoV-2 ORF8 Forms Intracellular Aggregates and Inhibits IFNγ-Induced Antiviral Gene Expression in Human Lung Epithelial Cells. Frontiers in Immunology, 12, 679482.

Publication 2021

RIPA lysis buffer Halt protease inhibitor cocktail Pierce BCA protein assay kit 4-20% TGX precast SDS-PAGE gels PVDF membranes

Actin Assay Buffer Cell Gels Immunoblot analyses Membranes Monoclonal antibody Mouse Protease inhibitor Protein Pvdf Ripa Sds page

Corresponding Organization : Jesse Brown VA Medical Center

Other organizations : University of Chicago

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Expression of GFP protein

control variables

Total protein concentration was kept at 20 µg per sample
β-actin levels were used as a loading control

controls

Positive control: None specified
Negative control: None specified

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