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Testing where in the cell Lyn is.  This test may be a debug, if it doesn't localize, if may be because it doesn't need to associate with CD79A/B for very long to phosphorylate it, it may drift over to CD79A/B, phosphorylate it, then leave.  A failure at this test may indicate the reason for a failure of Syk localization, but it does not indicate a problem with the BCR on its own.

To determine if our circuit is assembling the way we expect it to and to see if Lyn recruitment is dependent on activation (which could mean that Lyn recruitment be used as a mechanism for signal transduction).

BCR (CD79A/B, IgM heavy, IgM light), Lyn-mKate

Plasmidpurpose

pEXPR hEF1a: CD79A+STOP

 

pEXPR hEF1a: CD79B+STOP

 

pEXPR hEF1a: Gmab Heavy

 

pEXPR hEF1a: Gmab Light

 

pEXPR TRE: Lyn-mKate

 
hef1a:eBFPtransfection marker

 

anti IgM

Well 1

HEK293

 

Well 2

HEK293

Lyn-mKate

Dummy DNA

Well 3

HEK293

GmabH

GmabL

CD79A

CD79B

Lyn-mKate

Well 4

EMPTY

Well 5 

EMPTY

 

Well 6 

EMPTY

 

Well 7

HEK293

Oligomerized Beta Amyloid

Well 8 

HEK293

Lyn-mKate

Dummy DNA

Oligomerized Beta Amyloid

Well 9 

HEK293

GmabH

GmabL

CD79A

CD79B

Lyn-mKate

Oligomerized Beta Amyloid (conc. 1)

Well 10 

HEK293

GmabH

GmabL

CD79A

CD79B

Lyn-mKate

Oligomerized Beta Amyloid (conc. 2)

Well 11 

EMPTY

 

Well 12 

EMPTY

 

Well 13

HEK293

Monomerized Beta Amyloid

Well 14

HEK293

Lyn-mKate

Dummy DNA

Monomerized Beta Amyloid

Well 15

HEK293

GmabH

GmabL

CD79A

CD79B

Lyn-mKate

Monomerized Beta Amyloid (conc. 1)

Well 16

HEK293

GmabH

GmabL

CD79A

CD79B

Lyn-mKate

Monomerized Beta Amyloid (conc. 2)

Well 17

EMPTY


Well 18

EMPTY


Well 19

HEK293

Anti IgM

Well 20

HEK293

Lyn-mKate

Dummy DNA

Anti IgM

Well 21

HEK293

GmabH

GmabL

CD79A

CD79B

Lyn-mKate

Anti IgM (conc. 1)

Well 22

HEK293

GmabH

GmabL

CD79A

CD79B

Lyn-mKate

Anti IgM (conc. 2)

Well 23

EMPTY


Well 24

EMPTY


Lyn should localize in at least some samples. Hopefully Lyn will only localize if the BCR is active and will not localize if it is inactive.

 

 

 

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