The assay presented here was designed to assess the immediate effects of ethanol (EtOH) exposure on intracellular signaling activated by BMPs (Bone Morphogenetic Proteins). Previous reports of the relationship between EtOH exposure and BMP-dependent signaling have primarily assessed the expression of individual BMPs, changes in BMP target genes or effects on the phosphorylation level of key downstream mediators after days or weeks of in vivo EtOH exposure. What happens to BMP-stimulated signaling immediately following exposure to EtOH remains largely unexplored. Here, the early events of BMP-evoked intracellular signaling were examined in an in vitro model of acute EtOH toxicity. The BMP/Ethanol Stimulation Assay involved first stimulating cultured cells with recombinant BMPs. BMP-evoked intracellular signaling was then allowed to develop for 30 minutes. Next, the cells were exposed to a range of EtOH concentrations for an additional 30 minutes. Finally, the cultures were processed for Western blot analysis or immunofluorescent labeling. This short-term assay: • Permits investigation of EtOH exposure during the initial signaling events downstream of BMP receptor activation • Enables assessment of how the presence of BMPs might protect against cellular injury caused by toxic EtOH levels.
Keywords: BMPs, Bone Morphogenetic Proteins; C2C12 cells; CGM, Complete Growth Medium; DRG neurons; DRG, Dorsal Root Ganglion; EtOH, Ethanol; IF, Immunofluorescence; Immunofluorescent labeling; PAE, Prenatal Alcohol Exposure; PI3K, Phosphatidylinositol 3-kinase; PI3K/Akt; PS, Penicilin/Streptomycin; PSG, Penicilin/Strepomycin/Glutamine; SSM, Serum starvation medium; Smad; Western blot.
© 2022 The Author(s).