Western Blot Analysis of Fli-1 Expression

Cells were lysed in Radioimmunoprecipitation assay (RIPA) lysis buffer. After lysis, the supernatant was collected. Equal amounts of protein (20 µg-40 µg) were run for 1.5 hours at 130V on a 4–20% Criterion TGX Stain-Free Protein Gel (Bio-Rad, Hercules, CA) and electrotransferred to a PVDF membrane by Iblot2 transfer stacks (Invitrogen, Waltham, MA). Transferred proteins were probed with both a Fli-1 polyclonal antibody described previously (20 (link)) and an antibody to β-actin (Cell Signaling, Beverly, MA). The results were visualized using the Odyssey Imaging System (LI-COR, Lincoln, NE).

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Wang X., Richard M.L., Caldwell T.S., Sundararaj K., Sato S., Nowling T.K, & Zhang X.K. (2023). Role of the transcription factor Fli-1 on the CXCL10/CXCR3 Axis*. Frontiers in Immunology, 14, 1219279.

Publication 2023

4–20% Criterion TGX Stain-Free Protein Gel Iblot2 transfer stacks antibody to β-actin Odyssey Imaging System

Actin Antibody Buffer Cells Membrane Protein a Proteins Pvdf Radioimmunoprecipitation assay Stain

Corresponding Organization : Medical University of South Carolina

Other organizations : Xiangya Hospital Central South University, Central South University, Fukushima Medical University

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Variable analysis

independent variables

Protein lysis buffer (RIPA buffer)

dependent variables

Protein expression levels of Fli-1 and β-actin

control variables

Amount of protein loaded (20 µg-40 µg)
Gel run time (1.5 hours)
Gel voltage (130V)
Gel type (4–20% Criterion TGX Stain-Free Protein Gel)
Transfer method (Iblot2 transfer stacks)
Antibodies used (Fli-1 polyclonal antibody and β-actin antibody)
Visualization method (Odyssey Imaging System)

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