Group A: Membrane Localization
In this experiment, we want to test wether or not the receptor proteins, LilrB2 and PirB, localize to the membrane when exogenously expressed in cells.
Group B: Receptor Affinity for Beta-amyloid
In this experiment, we want to test that beta-amyloid binds to the receptor proteins that we have introduced to the cell. We may potentially extend this experiment to trying to characterize the interaction between beta-amyloid and the receptors i.e. try to generate saturation plots and calculate dissociation constants.
Group C: Levels of Endogenous Cofilin
comparing endogenous Syk expression to expression of some protein under TRE, since in theory the expression level of TRE:(random protein) should reflect the amount of Syk-TEVp we'll get out from TRE:Syk-TEVp. What Brian suggested was that the "random protein" we use should actually be Syk-TEVp or Syk-eYFP (I guess it removes some of the variables). This way we can compare endogenous and exogenous Syk production directly. In a Western blot, this involves essentially using the antibodies to stain two different proteins - the native Syk and a Syk-fusion. Since Syk and Syk-fusion are different sizes, there should be two different bands on the Western blot afterwards, whose strength we can compare. I'm not sure whether we'll end up using Syk-eYFP or Syk-TEVp for our blots but I can't imagine that it matters all that much. (Maybe we'll try out both?)
Group D: Cofilin Recruitment
- Find data on receptor-cofilin dynamics (esp. how long cofilin stays recruited at the membrane)
- pEXPR hEF1a:cofilin-eYFP
- look at structure of cofilin to determine which (N or C) terminus to put the fluorescent protein
- order cofilin gBlock
- look into different types of cofilin
- look for higher intensity at around membrane
- pEXPR hef1a:eBFP
Group E: (Whole System) Cofilin + TEV Protease Recruitment to Receptor + Cleavage Site + Transcription Factor
- pEXPR hEF1a:Lilrb2-TCS-Gal4VP16
- pEXPR TRE:cofilin-TEV
- pEXPR Gal4UAS:eYFP
- pEXPR hEF1a:rtTA3
- pEXPR hEF1a:eBFP transfection marker
- pEXPR TRE:mKate indicator of effect of dox on transcription
How much TEV Protease we need in the system? - indicated by levels of dox
looking for biggest difference in transfer curve between w/ AB and without AB
two different ladders - dox and AB