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