CFTR research paper Flashcards
What does syntaxin-1A do?
Interacts with CFTR and inhibits channel activity
Where is synatxin-8 found?
In foetal lung library- DNA found there
What is the aim of the paper?
To see if syn-8 regulates CFTR and if so how
What was the first thing they did?
Made CHO-CFTR and CHO-2T which were over expressed cells and CFTR+syn8 over expressed cells
How did they check that the over expressed cells had worked?
Western blotting- showed CHO-CFTR had a bit of endogenous syn-8 but CHO-2T had over expressed
Immunoprecipitation- check CFTR is present in both
What did they do with Iodide?
Loaded cells with radioactive Iodide and activated CFTR with Forskolin and saw how quickly Iodide left the cell- much quick without syn-8
What is syn8TM?
mutant Syn8 that lacks anchor
What showed that normal syn8 is needed to regulate CFTR not mutant?
Fluorescent tagged ABs of CFTR- show CFTR not at membrane with wild type but is with mutant
Iodide with mutant and wildtype
What else other than the Iodide shows that syn8 effects CFTR?
Patch clamp- measured whole cell CFTR currents with CHO-CFTR and CHO-2T
What showed the location of CFTR, CFTR and syn8, CFTR and syn8TM?
GFP-CFTR, red-syn8, yellow- colocalised
What was critical analysis the fluorescence showing the location of each?
Does the final image actually show CFTR+syn8 is on plasma membrane
What study shows where the co-localisation sites are within the cell?
Compared the CFTR and syn8 expression markers flourescence with Lamp1 (no), transferrin (bit)receptors, Rab11 (a lot)- so a lot in Golgi
What study showed whether there was actual physical interaction of CFTR and syn8?
immunoprecepitation - AB pull out protein and anything thats attached
showed CFTR pulled out syn8 and vice versa
What was the in vitro affinity binding assay?
GST fusion protein where syn1ATm, syn8TM, syn8[1-99], syn8[99-209] were stuck to beads and put into serrate column, CFTR was flushed through and can see what sticks to by western blot.
Showed strong stick to syn1A so direct binding and weak stick to syn8 so indirect binding
What were the conclusion of the experiment and why?
Syn8 inhibits CFTR function - patch and iodide
Syn8 affects trafficking of CFTR- co-localisation experiment, reduce CFTR from membrane
Syn8 must be anchored to membrane - co-localisation - syn8TM lack membrane tether
Syn8 and CFTR got to recycling endoscopes and TGN membranes- colocalisation with Rab11 and transferrin
Direct interaction syn8 and CFTR- immunoprecipitation and in vitro binding assay
