Western Analysis of Breast Cancer 1 Protein (BRCA1)

The breast cancer 1 protein ( BRCA1 ) facilitates DNA repair, preventing embryolethality and protecting the fetus from reactive oxygen species ( ROS )-induced developmental disorders mediated by oxidatively damaged DNA. Alcohol (ethanol, EtOH ) exposure during pregnancy causes fetal alcohol spectrum disorders ( FASD ), characterized by aberrant behaviour and enhanced ROS formation and proteasomal protein degradation. Herein, ROS-producing NADPH oxidase ( NOX ) activity was higher in Brca1 +/− vs. +/+ fetal and adult brains, and further enhanced by a single EtOH exposure. EtOH also enhanced catalase and proteasomal activities, while conversely reducing BRCA1 protein levels without affecting Brca1 gene expression. EtOH-initiated adaptive postnatal freezing behaviour was lost in Brca1 +/− progeny. Pretreatment with the free radical spin trap and ROS inhibitor phenylbutylnitrone blocked all EtOH effects, suggesting ROS-dependent mechanisms. This is the first in vivo evidence of NOX regulation by BRCA1, and of EtOH-induced, ROS-mediated depletion of BRCA1, revealing novel mechanisms of BRCA1 protection in FASD.

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“…BRCA1 protein levels were measured via western blotting performed as described in Drake et al [ 46 ], and detailed in the supplementary data.…”

Section: Methodsmentioning

confidence: 99%