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Flashcards in L8 - Critcal Paper Review Deck (37)
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1

Author and date

Bilan et al. 2004

2

CFTR interacts with protein which contain what domain

PDZ

3

What general class of proteins are involved in vesicle fusion and trafficking

SNARe proteins

4

Syntaxin belongs to which family of proteins

SNARE

5

What is the effect of interaction of syntaxin 1A and CFTR

Inhibition of channel activity

6

What cell lines were produced

cDNA Syn8, Syn1-A, Syn3 cell lines
cDNA CFTR lines
Mutant syn8 delta TM

7

What was mutated in Syn8deltaTM

Lacked the transmembrane anchor - so was unable to anchor into the cell membrane

8

What was CFTR fused with

GFP

9

How many forms of CFTR, why?

Two
One mature, the other lack glycosylation

10

What is Fsk, what was its effect

Forskolin
Activation of CFTR

11

What were the methods for the Iodide eflux experiemnt

Load cells with radioactive iodide
Look at how quickly it leaves the cells - out through CFTR

12

What were the results and conclusions from the iodide eflux

With Syn8 rate of I loss was less therefore due to either a change in number of CFTR channels at the membrane or change in Po

13

In the immunoflourescence experiement where was
CFTR
Syn8
Syn8deltaTM

Membrane
Membrane
Cytosol

14

What were the conclusions from this immunoflourescence experiement

Syn8 inhibits the response of CFTR to FSK but this can only be achieved if Syn8 is in the membrane

15

What information does a whole cell patch clamp give

Total currents due to ion channel function

16

In a whole cell patch clamp the further a value is away from 0 the ...

Larger the ion channel function

17

What were the results of the whole cell patch clamp - what was the effect of syn8

Decreased CFTR magnitude

18

What is the effect of Syn8 on the trafficking of CFTR
What is the effect of Syn8deltaTM on the trafficking of CFTR
What experiement was this seen in

Inhibits
No effect
Immunoflourescence

19

Lysosome markers

Lamp1

20

Was their co-localisation of CFTR and Syn8 in the lysosomes

No

21

Endocytic recycling marker

Transferrin

22

Was their co-localisation of CFTR and Syn8 in endocytic recylcing vesicles

Yes, a little

23

Recycling/post golgi marker

Rab11

24

Was there co-localisation of CFTR and Syn8 in Recycling/post golgi

Yes

25

What experiement was carried out to test for physical interaction

Immunoprecipitation

26

What happens in a Immunoprecipitation

An antibody is used to pull out target protein, any other proteins that are attached are also pulled out

27

What happened in the Immunoprecipitation when a Syn8 ab was used

Syn8 AB pulled out Syn8 and CFTR

28

What happened in the Immunoprecipitation when a CFTR ab was used

CFTR AB pulled out CFTR and Syn8

29

What was the second experiment used to look for physical interaction

In vitro binding assay

30

Explain how an in vitro binding assay works

Bead bound GST fusion protein incubated in cell lysate
Protein that binds to the beads examined by Western blot