You are studying a genetic disease where patients have muscle weakness due to muscle cells failing to properly proliferate and fuse together into mature fibers. You know that muscle cells should respond to growth factors that activate different signaling cascades, which will increase the transcription of different genes.
In normal muscle cells, the Wnt-B catenin pathway is necessary for the proliferation of myoblasts. Wnt (the ligand) will bind to Fzd (G-protein coupled receptor) and set off a cascade that allows B catenin to go into the nucleus and turn on transcription of genes such as CREB, ATF, and Lef1.
You want to determine if patients have a defect in Wnt signaling or another pathway. If there is a defect in Wnt-signaling, you want to determine if the defect is due to a mutant protein in the signaling cascade, a mutant B catenin that reduces DNA binding, or in a mutation in a gene that reduces expression of genes that should be turned on by Wnt-B catenin signaling.
What type of experiment would determine if there was a problem with Wnt binding to the Fzd receptor, or if there was a problem with the Fzd receptor initiating the proper signaling cascade?
Group of answer choices
Treat muscle cells with Wnt and then lyse cells using native cell lysis buffer. Test the lysate for G-protein activity. If the lysate can activate kinases in the Wnt-B catenin pathway, the G-protein was bound to GTP and was activated by Wnt binding to the Fzd receptor.
Treat muscle cells with Wnt and then lyse cells using an SDS buffer. Test the lysate for G-protein activity. If the lysate can phosphorylate B catenin, then the G-protein was bound to GTP and was activataed by Wnt binding to the Fzd receptor.
Treat muscles cells with Wnt and then crosslink proteins. Test the lysate for Fzd receptor activity. If the Fzd receptor is bound to GTP, then Fzd receptor was activated by the Wnt ligand.
Treat muscle cells with Wnt and then crosslink proteins. Use an co-immunoprecipitation assay and an antibody that binds the Wnt peptide. Run the sample on a Western Blot and blot with an antibody against Fzd. If there is a band for Fzd on the Western Blot, then Wnt was able to bind the Fzd receptor.