Western Blot Analysis of Viral Proteins

Whole-cell lysates were subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (pore size 0.45 µm, Merck Millipore, MA, USA). PVDF membranes were blocked with 5% skim milk or Blocking One (Nacalai tesque, Kyoto Japan) in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) and probed with anti-Tax mouse monoclonal (Lt-4), anti-HBZ mouse monoclonal (#7-1) [23 (link)], anti-HBZ rat monoclonal (#4B12), anti-β-actin rabbit polyclonal (PM053, MEDICAL & BIOLOGICAL LABORATORIES, Nagoya, Japan), or anti-α-tubulin rabbit polyclonal (PM054, MEDICAL & BIOLOGICAL LABORATORIES) antibodies. PVDF membranes were washed with TBS-T and incubated with IRDye 680RD Goat anti-Mouse IgG, IRDye 800CW goat anti-rat IgG, or IRDye 800CW goat anti-rabbit IgG (LI-COR Biosciences, Lincoln, NE). After washing with TBS-T, the proteins were detected using Odyssey CLx Infrared Imaging System (LI-COR Biosciences).

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Naito T., Yasunaga J.I., Mitobe Y., Shirai K., Sejima H., Ushirogawa H., Tanaka Y., Nakamura T., Hanada K., Fujii M., Matsuoka M, & Saito M. (2018). Distinct gene expression signatures induced by viral transactivators of different HTLV-1 subgroups that confer a different risk of HAM/TSP. Retrovirology, 15, 72.

Publication 2018

PVDF) membranes Blocking One IRDye 680RD Goat anti-Mouse IgG IRDye 800CW goat anti-rat IgG IRDye 800CW goat anti-rabbit IgG Odyssey CLx Infrared Imaging System

Actin Anti igg Antibodies Biological Cell Goat Irdye 800cw Membranes Milk Mouse Polyvinylidene difluoride Proteins Rabbit Saline Sds polyacrylamide gel electrophoresis Tween 20 α tubulin

Corresponding Organization :

Other organizations : Kawasaki Medical School, Kyoto University, Kyushu Institute of Technology, University of the Ryukyus, Nagasaki International University, Niigata University, Kumamoto University

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Variable analysis

independent variables

Treatment with anti-Tax mouse monoclonal (Lt-4) antibody
Treatment with anti-HBZ mouse monoclonal (#7-1) antibody
Treatment with anti-HBZ rat monoclonal (#4B12) antibody

dependent variables

Protein expression levels of Tax, HBZ, β-actin, and α-tubulin

control variables

SDS-PAGE and transfer of whole-cell lysates to PVDF membranes
Blocking of PVDF membranes with 5% skim milk or Blocking One
Washing of PVDF membranes with Tris-buffered saline containing 0.1% Tween 20 (TBS-T)
Incubation of PVDF membranes with secondary antibodies (IRDye 680RD Goat anti-Mouse IgG, IRDye 800CW goat anti-rat IgG, or IRDye 800CW goat anti-rabbit IgG)
Detection of proteins using Odyssey CLx Infrared Imaging System

positive controls

None specified

negative controls

None specified

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