Description:
In this experiment, we are trying to asses the extent of the cleavage of the TCS fused to the receptors by TEVp-Cofilin given different levels of expression of TEVp-Cofilin. Cleavage of the TCS will result in the release of the transcription factor Gal4VP16 which will in turn activate the Gal4UAS driven mKate. We also want to assess the amount of background activation of Gal4VP16:mKate
We have reason to believe that there are high levels of endogenous cofilin expression in HEK293 cells (cofilin is used as a loading control fro Western blots, lol). The levels of endogenous vs exogenous cofilin will be assessed through the cofilin Western blot experiment. However, if this is the case, we don't expect to see any mKate Gal4UAS activation at low levels of expression of TEVp-Cofilin as the endogenous cofilin is likely to overwhelm the exogenous TEVp-Cofilin. Hopefully, we do get an increase in mKate expression at some high level of dox induction. This may indicate that we could just express TEVp-Coflin constitutively.
LilrB2:
PirB:
Trial 1
We are doing the first PirB Trial without using beta-amyloid to activate the receptor, since we are not actually sure whether or not it binds the receptor.
Trial 2
Results
LilrB2
| Well 1 | Well 2
| Well 3
| Well 4
| Well 5
| |
Well 7
| Well 8
| Well 9
| Well 10 
| Well 11
| Well 12
|
Well 1HEK293 | Well 2HEK293 hEF1a:eBFP 200ng hEF1a:PirB 200ng hEF1a:rtTA 200ng TRE:TEVp-Cofilin 200ng Dummy 200ng | Well 3HEK293 hEF1a:eBFP 200ng hEF1a:PirB 200ng hEF1a:rtTA 200ng TRE:TEVp-Cofilin 200ng Gal4UAS:mKate 200ng | Well 4HEK293 hEF1a:eBFP 200ng hEF1a:PirB-TCS-Gal4VP16 200ng hEF1a:rtTA 200ng Gal4UAS:mKate 200ng Dummy 200ng | Well 5HEK293 hEF1a:eBFP 200ng hEF1a:Gal4VP16 200ng Gal4UAS:mKate 200ng Dummy 400ng | |
|
|
|
|
| |
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 100nM DOX | Well 11HEK293 hEF1a:eBFP 200ng hEF1a:PirB-TCS-Gal4VP16 200ng hEF1a:rtTA 200ng TRE:TEVp-Cofilin 200ng Gal4UAS:mKate 200ng 1000nM DOX | Well 12HEK293 hEF1a:eBFP 200ng hEF1a:PirB-TCS-Gal4VP16 200ng hEF1a:rtTA 200ng TRE:TEVp-Cofilin 200ng Gal4UAS:mKate 200ng 2000nM DOX |
|  |  |  |  |  |
PirB
Well 7
| Well 8
| Well 9
| Well 10
| Well 11
| Well 12
|
Well 1HEK293 | Well 2HEK293 hEF1a:eBFP 200ng hEF1a:PirB 200ng hEF1a:rtTA 200ng TRE:TEVp-Cofilin 200ng Dummy 200ng | Well 3HEK293 hEF1a:eBFP 200ng hEF1a:PirB 200ng hEF1a:rtTA 200ng TRE:TEVp-Cofilin 200ng Gal4UAS:mKate 200ng | Well 4HEK293 hEF1a:eBFP 200ng hEF1a:PirB-TCS-Gal4VP16 200ng hEF1a:rtTA 200ng Gal4UAS:mKate 200ng Dummy 200ng | Well 5HEK293 hEF1a:eBFP 200ng hEF1a:Gal4VP16 200ng Gal4UAS:mKate 200ng Dummy 400ng | |
|
|
|
|
| |
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 100nM DOX | Well 11HEK293 hEF1a:eBFP 200ng hEF1a:PirB-TCS-Gal4VP16 200ng hEF1a:rtTA 200ng TRE:TEVp-Cofilin 200ng Gal4UAS:mKate 200ng 1000nM DOX | Well 12HEK293 hEF1a:eBFP 200ng hEF1a:PirB-TCS-Gal4VP16 200ng hEF1a:rtTA 200ng TRE:TEVp-Cofilin 200ng Gal4UAS:mKate 200ng 2000nM DOX |
|  |  |  |  |  |
Analysis
PirB
From the Well 4 control (all components without TEVp-Cofilin) we see that there is a significant amount of background activation of Gal4UAS:mKate. Interestingly, however, we do not see this background activation when TEVp-Cofilin is present but without any dox induction.
**It is interesting to note that eBFP and tagBFP give 'unusual' microscopy and cytometry data
these are evos pictures of the same population of cells expressing mKate and tagBFP constitutively under the blue channel and the red channel
it is important to keep this in mind when checking for transfection efficiency on the evos of cells expressing a blue fluorescent protein as a transfection marker
| red channel | blue channel |
 |  |