Group E: Testing TEVp-Cofilin cleavage with active receptors

Created by Unknown User (lylaatta@mit.edu), last modified on Sep 25, 2014 11:39

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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.

LilrB2:

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:

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*

	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

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