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Description:

In this experiment, we

want to test for the recruitment of our engineered cofilin to the activated receptor proteins. To do this, we will transfect HEK293 cells with LilrB2 and PirB as well as coflin-eYFP under a TRE promoter. We will then apply beta-amyloid oligomers in order to activate the receptors and add varying amounts of dox to induce varying levels of expression of cofilin-eYFP. We will look for increased yellow light intensity near the membrane of the cell. We test different levels of expression of cofilin because we don't know how the levels of endogenous cofilin in the cell will affect the interaction between our receptor and our engineered cofilin. We are looking for the level of expression of cofilin that will result in a discernible increased intensity of yellow near the membrane, indicating that a significant amount of cofilin-eYFP has recruited to the membrane.

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.

LilrB2:

Expand

title	LilrB2 Trial 1

Well 1

HEK293

Well 2

HEK293

hEF1a:eBFP 200ng

hEF1a:LilrB2 200ng

hEF1a:rtTA 200ng

TRE:TEVp-Cofilin200ng

Well 3

HEK293

hEF1a:eBFP 200ng

hEF1a:LilrB2 200ng

hEF1a:rtTA 200ng

TRE:TEVp-Cofilin200ng

Well 4

HEK293

hEF1a:eBFP 200ng

hEF1a:LilrB2-TCS-Gal4VP16 200ng

hEF1a:rtTA 200ng

Gal4UAS:mKate200ng

Dummy 200ng

Well 5

HEK293

hEF1a:eBFP 200ng

hEF1a:LilrB2-TCS-Gal4VP16 200ng

hEF1a:rtTA 200ng

Gal4UAS:mKate200ng

Dummy 200ng

500 nM aBeta

Well 7

HEK293

hEF1a:eBFP 200ng

hEF1a:LilrB2-TCS-Gal4VP16 200ng

hEF1a:rtTA 200ng

TRE:TEVp-Cofilin 200ng

Gal4UAS:mKate 200ng

0nM DOX

Well 8

HEK293

hEF1a:eBFP 200ng

hEF1a:LilrB2-TCS-Gal4VP16 200ng

hEF1a:rtTA 200ng

TRE:TEVp-Cofilin 200ng

Gal4UAS:mKate 200ng

1nM DOX

Well 9

HEK293

hEF1a:eBFP 200ng

hEF1a:LilrB2-TCS-Gal4VP16 200ng

hEF1a:rtTA 200ng

TRE:TEVp-Cofilin 200ng

Gal4UAS:mKate 200ng

10nM DOX

Well 10

HEK293

hEF1a:eBFP 200ng

hEF1a:LilrB2-TCS-Gal4VP16 200ng

hEF1a:rtTA 200ng

TRE:TEVp-Cofilin 200ng

Gal4UAS:mKate 200ng

1000nM DOX

Well 11

Well 13

HEK293

hEF1a:eBFP 200ng

hEF1a:LilrB2-TCS-Gal4VP16 200ng

hEF1a:rtTA 200ng

TRE:TEVp-Cofilin 200ng

Gal4UAS:mKate 200ng

0nM DOX

500 nM aBeta

Well 14

HEK293

hEF1a:eBFP 200ng

hEF1a:LilrB2-TCS-Gal4VP16 200ng

hEF1a:rtTA 200ng

TRE:TEVp-Cofilin 200ng

Gal4UAS:mKate 200ng

1nM DOX

500 nM aBeta

Well 15

HEK293

hEF1a:eBFP 200ng

hEF1a:LilrB2-TCS-Gal4VP16 200ng

hEF1a:rtTA 200ng

TRE:TEVp-Cofilin 200ng

Gal4UAS:mKate 200ng

10nM DOX

500 nM aBeta

Well 16

HEK293

hEF1a:eBFP 200ng

hEF1a:LilrB2-TCS-Gal4VP16 200ng

hEF1a:rtTA 200ng

TRE:TEVp-Cofilin 200ng

Gal4UAS:mKate 200ng

10000nM DOX

500 nM aBeta

PirB:

Expand

title	PirB Trial 1

Well 1

HEK293

Well 2

HEK293

hEF1a:eBFP 200ng

hEF1a:PirB 200ng

hEF1a:rtTA 200ng

TRE:TEVp-Cofilin200ng

Well 3

HEK293

hEF1a:eBFP 200ng

hEF1a:PirB 200ng

hEF1a:rtTA 200ng

TRE:TEVp-Cofilin200ng

Well 4

HEK293

hEF1a:eBFP 200ng

hEF1a:PirB-TCS-Gal4VP16 200ng

hEF1a:rtTA 200ng

Gal4UAS:mKate200ng

Dummy 200ng

Well 5

HEK293

hEF1a:eBFP 200ng

hEF1a:PirB-TCS-Gal4VP16 200ng

hEF1a:rtTA 200ng

Gal4UAS:mKate200ng

Dummy 200ng

500 nM aBeta

Well 7

HEK293

hEF1a:eBFP 200ng

hEF1a:PirB-TCS-Gal4VP16 200ng

hEF1a:rtTA 200ng

TRE:TEVp-Cofilin 200ng

Gal4UAS:mKate 200ng

0nM DOX

Well 8

HEK293

hEF1a:eBFP 200ng

hEF1a:PirB-TCS-Gal4VP16 200ng

hEF1a:rtTA 200ng

TRE:TEVp-Cofilin 200ng

Gal4UAS:mKate 200ng

1nM DOX

Well 9

HEK293

hEF1a:eBFP 200ng

hEF1a:PirB-TCS-Gal4VP16 200ng

hEF1a:rtTA 200ng

TRE:TEVp-Cofilin 200ng

Gal4UAS:mKate 200ng

10nM DOX

Well 10

HEK293

hEF1a:eBFP 200ng

hEF1a:PirB-TCS-Gal4VP16 200ng

hEF1a:rtTA 200ng

TRE:TEVp-Cofilin 200ng

Gal4UAS:mKate 200ng

1000nM DOX

Well 11

Well 13

HEK293

hEF1a:eBFP 200ng

hEF1a:PirB-TCS-Gal4VP16 200ng

hEF1a:rtTA 200ng

TRE:TEVp-Cofilin 200ng

Gal4UAS:mKate 200ng

0nM DOX

500 nM aBeta

Well 14

HEK293

hEF1a:eBFP 200ng

hEF1a:PirB-TCS-Gal4VP16 200ng

hEF1a:rtTA 200ng

TRE:TEVp-Cofilin 200ng

Gal4UAS:mKate 200ng

1nM DOX

500 nM aBeta

Well 15

HEK293

hEF1a:eBFP 200ng

hEF1a:PirB-TCS-Gal4VP16 200ng

hEF1a:rtTA 200ng

TRE:TEVp-Cofilin 200ng

Gal4UAS:mKate 200ng

10nM DOX

500 nM aBeta

Well 16

HEK293

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 1 HEK293	Well 2 HEK293 hEF1a:eBFP 200ng hEF1a:PirB 200ng hEF1a:rtTA 200ng TRE:TEVp-Cofilin200ng	Well 3 HEK293 hEF1a:eBFP 200ng hEF1a:PirB 200ng hEF1a:rtTA 200ng TRE:TEVp-Cofilin200ng	Well 4 HEK293 hEF1a:eBFP 200ng hEF1a:PirB-TCS-Gal4VP16 200ng hEF1a:rtTA 200ng Gal4UAS:mKate200ng Dummy 200ng	Well 5 HEK293 hEF1a:eBFP 200ng hEF1a:PirB-TCS-Gal4VP16 200ng hEF1a:rtTA 200ng Gal4UAS:mKate200ng Dummy 200ng *500 nM aBeta*
	Image Added	Image Added	Image Added	Image Added	Image Added
	0 nM Dox	1 nM Dox	10 nM Dox	1000 nM Dox
- aBeta	Image Added	Image Added	Image Added	Image Added
+ aBeta	Image Added	Image Added	Image Added	Image Added

Means:

No TEvp-Cofilin Transfected

- aBeta

+ aBeta

Mean: 5827

Image Added

Mean: 6840

Image Added

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

Image Added

Mean: 8750

Image Added

Mean: 8707

Image Added

Mean: 8020

Image Added

+ aBeta

Mean: 8861

Image Added

Mean: 8037

Image Added

Mean: 8108

Image Added

Mean: 7988

Image Added

* We can potentially test for the recruitment of cofilin to the receptors through co-immunoprecipitation.