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Description:
In this experiment, we are testing whether or not beta amyloid activates the receptors, recruiting TEVp-Cofilin and leading to higher TCS cleavage and in turn, higher Gal4UAS activation. From experiment D, we determined that the level of TEVp-Cofilin expression corresponding to 0 nM dox was the level at which there was the least background TCS cleavage. We are, therefore, using TRE:TEVp-Cofilin with no dox induction in our experiment.
PirB:
PirB Trial 1
Well 1HEK293 | Well 2HEK293 hEF1a:eBFP 200ng hEF1a:PirB 200ng hEF1a:rtTA 200ng TRE:TEVp-Cofilin200ng
| Well 3HEK293 hEF1a:eBFP 200ng hEF1a:PirB 200ng hEF1a:rtTA 200ng TRE:TEVp-Cofilin200ng | Well 4HEK293 hEF1a:eBFP 200ng hEF1a:PirB-TCS-Gal4VP16 200ng hEF1a:rtTA 200ng Gal4UAS:mKate200ng Dummy 200ng | Well 5HEK293 hEF1a:eBFP 200ng hEF1a:PirB-TCS-Gal4VP16 200ng hEF1a:rtTA 200ng Gal4UAS:mKate200ng Dummy 200ng 500 nM aBeta
|
Well 7HEK293 hEF1a:eBFP 200ng hEF1a:PirB-TCS-Gal4VP16 200ng hEF1a:rtTA 200ng TRE:TEVp-Cofilin 200ng Gal4UAS:mKate 200ng 0nM DOX | Well 8HEK293 hEF1a:eBFP 200ng hEF1a:PirB-TCS-Gal4VP16 200ng hEF1a:rtTA 200ng TRE:TEVp-Cofilin 200ng Gal4UAS:mKate 200ng 1nM DOX | Well 9HEK293 hEF1a:eBFP 200ng hEF1a:PirB-TCS-Gal4VP16 200ng hEF1a:rtTA 200ng TRE:TEVp-Cofilin 200ng Gal4UAS:mKate 200ng 10nM DOX | Well 10HEK293 hEF1a:eBFP 200ng hEF1a:PirB-TCS-Gal4VP16 200ng hEF1a:rtTA 200ng TRE:TEVp-Cofilin 200ng Gal4UAS:mKate 200ng 1000nM DOX | Well 11 |
Well 13HEK293 hEF1a:eBFP 200ng hEF1a:PirB-TCS-Gal4VP16 200ng hEF1a:rtTA 200ng TRE:TEVp-Cofilin 200ng Gal4UAS:mKate 200ng 0nM DOX 500 nM aBeta | Well 14HEK293 hEF1a:eBFP 200ng hEF1a:PirB-TCS-Gal4VP16 200ng hEF1a:rtTA 200ng TRE:TEVp-Cofilin 200ng Gal4UAS:mKate 200ng 1nM DOX 500 nM aBeta | Well 15HEK293 hEF1a:eBFP 200ng hEF1a:PirB-TCS-Gal4VP16 200ng hEF1a:rtTA 200ng TRE:TEVp-Cofilin 200ng Gal4UAS:mKate 200ng 10nM DOX 500 nM aBeta | Well 16HEK293 hEF1a:eBFP 200ng hEF1a:PirB-TCS-Gal4VP16 200ng hEF1a:rtTA 200ng TRE:TEVp-Cofilin 200ng Gal4UAS:mKate 200ng 10000nM DOX 500 nM aBeta | |
Results:
PirB:
| Controls | Well 1HEK293 | Well 2HEK293 hEF1a:eBFP 200ng hEF1a:PirB 200ng hEF1a:rtTA 200ng TRE:TEVp-Cofilin200ng
| Well 3HEK293 hEF1a:eBFP 200ng hEF1a:PirB 200ng hEF1a:rtTA 200ng TRE:TEVp-Cofilin200ng | Well 4HEK293 hEF1a:eBFP 200ng hEF1a:PirB-TCS-Gal4VP16 200ng hEF1a:rtTA 200ng Gal4UAS:mKate200ng Dummy 200ng | Well 5HEK293 hEF1a:eBFP 200ng hEF1a:PirB-TCS-Gal4VP16 200ng hEF1a:rtTA 200ng Gal4UAS:mKate200ng Dummy 200ng 500 nM aBeta
|
 |  |  |  |  |
| 0 nM Dox | 1 nM Dox | 10 nM Dox | 1000 nM Dox | |
| - aBeta |  |  |  |  | |
| + aBeta |  |  |  |  | |
Means:
No TEvp-Cofilin Transfected
| - aBeta | + aBeta |
|---|
Mean: 5827 
| Mean: 6840 
|
Transfected with everything
Non blue population gated out
Red histogram shown; mean = mean red
| | 0 nM Dox | 1 nM Dox | 10 nM Dox | 1000 nM Dox |
| - aBeta | Mean: 9921 
| Mean: 8750 
| Mean: 8707 
| Mean: 8020 
|
| + aBeta | Mean: 8861 
| Mean: 8037 
| Mean: 8108 
| Mean: 7988 
|