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

 

anti IgM

Well 1

HEK293

 

Well 2

HEK293

hEF1a: eBFP

Dummy DNA

Well 3

HEK293

hEF1a: eBFP

hEF1a: rtTA

TRE: Lyn-mKate

Dummy DNA

0nM dox

Well 4

HEK293

hEF1a: eBFP

hEF1a: rtTA

TRE: Lyn-mKate

Dummy DNA

2000nM dox

Well 5 

 

Well 6 

 

Well 7

HEK 293

Anti-IgM

Well 8

HEK293

hEF1a: eBFP

Dummy DNA

Anti-IgM


Well 9

HEK293

hEF1a: eBFP

hEF1a: rtTA

TRE: Lyn-mKate

Dummy DNA

0nM dox

Anti-IgM

Well 10

HEK293

hEF1a: eBFP

hEF1a: rtTA

TRE: Lyn-mKate

Dummy DNA

2000nM dox

Anti-IgM

Well 11Well 12

Well 7

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A

CD79B

TRE: Lyn-mKate

0nM dox

Well 8

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A

CD79B

TRE: Lyn-mKate

1nM dox

Well 9

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A

CD79B

TRE: Lyn-mKate

10nM dox

Well 10

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A

CD79B

TRE: Lyn-mKate

100nM dox

Well 11

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A

CD79B

TRE: Lyn-mKate

1000nM dox

Well 12

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A

CD79B

TRE: Lyn-mKate

2000nM dox

Well 13

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A

CD79B

TRE: Lyn-mKate

0nM dox

Anti-IgM

Well 14

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A

CD79B

TRE: Lyn-mKate

1nM dox

Anti-IgM

Well 15

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A

CD79B

TRE: Lyn-mKate

10nM dox

Anti-IgM

Well 16

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A

CD79B

TRE: Lyn-mKate

100nM dox

Anti-IgM

Well 17

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A

CD79B

TRE: Lyn-mKate

1000nM dox

Anti-IgM

Well 18

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A

CD79B

TRE: Lyn-mKate

2000nM dox

Anti-IgM

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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