Silencing of Nrf genes in the human placenta as measured by SDS-PAGE and Western Blotting techniques

Yann W Yap; Natalie J Hannan; Euan M Wallace; Sarah A Marshall

doi:10.1016/j.placenta.2022.01.007

Silencing of Nrf genes in the human placenta as measured by SDS-PAGE and Western Blotting techniques

Placenta. 2022 Feb:118:70-74. doi: 10.1016/j.placenta.2022.01.007. Epub 2022 Jan 13.

Authors

Yann W Yap¹, Natalie J Hannan², Euan M Wallace³, Sarah A Marshall⁴

Affiliations

¹ The Hudson Institute of Medical Research, Clayton, 3168, Victoria, Australia; The Ritchie Centre, Department of Obstetrics and Gynaecology, School of Clinical Sciences, Monash University, Clayton, 3168, Victoria, Australia.
² Therapeutics Discovery and Vascular Function in Pregnancy Group, The Department of Obstetrics and Gynaecology, Mercy Hospital for Women, University of Melbourne & Mercy Perinatal, Mercy Hospital for Women, Heidelberg, 3084, Victoria, Australia.
³ The Ritchie Centre, Department of Obstetrics and Gynaecology, School of Clinical Sciences, Monash University, Clayton, 3168, Victoria, Australia.
⁴ The Hudson Institute of Medical Research, Clayton, 3168, Victoria, Australia; The Ritchie Centre, Department of Obstetrics and Gynaecology, School of Clinical Sciences, Monash University, Clayton, 3168, Victoria, Australia. Electronic address: sarah.marshall@monash.edu.

PMID: 35045361
DOI: 10.1016/j.placenta.2022.01.007

Abstract

Nuclear factor erythroid 2-related factor-2 (Nrf2), and the less well characterised proteins Nrf1 and Nrf3, are member of the cap 'n' collar family of transcription factors. Nrf proteins regulate the expression of endogenous antioxidant enzymes and have recently become the targets for various therapeutic treatments. Recently, Nrf proteins have been of particular interest as a target in placental-derived oxidative stress induced pregnancy disorders. Here, we report the presence of Nrf1, Nrf2 and Nrf3 proteins in both human primary trophoblast and human trophoblast choriocarcinoma cell line (BeWo). We also detail the steps taken to successfully silence all Nrf proteins in both human primary trophoblast cells and BeWo via detection of mRNA and protein using quantitative PCR, and SDS-PAGE and Western Blotting respectively.

Keywords: ARE; BeWo; Nrf1; Nrf2; Nrf3; Placenta; SDS-PAGE; Silencing; Trophoblast; Western blot; qPCR; siRNA.

Publication types

Evaluation Study
Research Support, Non-U.S. Gov't

MeSH terms

Basic-Leucine Zipper Transcription Factors / genetics
Basic-Leucine Zipper Transcription Factors / metabolism*
Blotting, Western
Cell Line, Tumor
Electrophoresis, Polyacrylamide Gel
Humans
NF-E2-Related Factor 2 / genetics
NF-E2-Related Factor 2 / metabolism
Primary Cell Culture
RNA Interference*
Trophoblasts / metabolism*

Substances

Basic-Leucine Zipper Transcription Factors
NF-E2-Related Factor 2
NFE2L2 protein, human