No point mutating Syk because we’re not going to worry about cross-talk
Talk to Nelson about maximum number plasmids for transfection
Neuronal crosstalk?
TEV cleavage site high or low affinity?
Experiment
Western blot HEK293 cells for Syk
- anti-Syk antibody
Check BCR membrane localization
- add CD79a/b + mIgM and verify with fluorescent anti-IgM antibodies
Check IgM binding
- biotinylated beta-amyloid with fluorescent secondary antibody
Check Lyn and Syk membrane localization using fluorescent fusions
- Lyn-YFP
- Syk-mKate
Make Syk+TEVp fusions
- Fusions -> Prioritize
- N terminus: 0 linking aa
- N terminus: 15 linking aa
- C terminus: 0 linking aa
- C terminus: 15 linking aa
- Inducible expression under TetR -> find upper limit for constitutive behavior
Syk+TEVp -> find lower limit of beta amyloid (threshold)
High Priority Fusions
- Syk-eYFP
- Lyn-mKATE
- CD79A-TCS-Gal4VP16
- CD79B-TCS-Gal4VP16
- Syk-15-TEVp
- (Syk-0-TEVp)
Use ENLYFQL cleavage site -> best on/off ratio
Add to C terminus of Syk like they did in paper with EGFP
Add to C terminus of Lyn because N terminus in membrane