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
In this experiment, we want to want to compare the levels of endogenous cofilin and cofilin-eYFP expressed under an inducible promoter with different levels of dox induction. This will allow us to determine what level of expression of cofilin would lead to the best signal:noise ratio of binding of exogenous cofilin. We are planning to transfect HEK293 with cofilin-eYFP under a TRE promoter and induce with varying levels of dox. We will then preform a Western blot with an anti-cofilin primary antibody and compare band sizes to test for the relative amounts of endogenous cofilin and exogenous cofilin-eYFP in the cells.
- Find data on receptor-cofilin dynamics (esp. how long cofilin stays recruited at the membrane)
- pEXPR TRE:cofilin-eYFP
- 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