Biomedical Engineering Reference
In-Depth Information
Fig. 5
Glycosidase treatment of surface biotinylated CFTR. Surface biotinylated
WT and
F508 CFTRs were treated with endoglycosidase H (Endo H), which
deglycosylates ER core glycosylation (band B) but not complex glycosylation
(band C). CFTRs were also treated with N-Glycosidase F (PNGase F), which
deglycosylates all N-glycan chains to remove carbohydrate residues from pro-
teins (band A). Data indicate that surface expressed WT-CFTR in control cells (
left
3 panels
) is resistant to Endo H. However, surface expressed WT (
middle 3 panels
)
or
ʔ
F508 (
right 3 panels
) CFTRs in cells treated with Arf1 mutant (Arf1-Q71L) are
sensitive to Endo H, indicating that they underwent only ER core glycosylation.
Modifi ed from Gee et al. [
3
] with permission
ʔ
2. Endo H treatment: Endo H is a recombinant glycosidase that
cleaves the chitobiose core of high mannose and some hybrid
oligosaccharides from N-linked glycoproteins. This conversion
takes place in the medial Golgi region. When proteins are cor-
rectly processed in the ER and travel through the Golgi, they
become resistant to Endo H. Therefore, sensitivity to Endo H
indicates the presence of proteins that have not been processed
beyond the ER.
Protocols: (a) Place 10-20
L
of 1× glycoprotein denaturing buffer. (b) Incubate the samples
at 100 °C for 10 min. Alternatively, for membrane proteins like
CFTR, protein samples can be incubated at 37 °C for 10 min
to avoid aggregation. (c) Add 2
ʼ
g of the protein samples in 10
ʼ
ʼ
L of 10 % NP-40, 2
ʼ
L of
10× Endo H reaction buffer, 1-5
L of Endo H, and enough
distilled water for a total reaction volume of 20
ʼ
L. Next, fur-
ther incubate the samples at 37 °C for 1-2 h. (d) Perform
SDS-PAGE and immunoblotting.
3. N-ethylmaleimide (NEM) treatment: NEM can be used to
detect NEM-sensitive factor attachment protein receptor
(SNARE)-mediated vesicular traffi cking, because NEM inhibits
membrane fusion by disrupting SNARE functions [
12
] (Fig.
6
).
Protocols: (a) Incubate cells at 20 °C for 2 h. (b) Treat cells
with NEM (1 mM) for 15 min at 20 °C. (c) Incubate cells in
serum-free media without NEM for 2 h at 37 °C. This step is
required to identify the effects of NEM on intracellular traf-
fi cking in comparison with control cells that had not been
ʼ
