JCS028951 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. CFTR chloride currents are dependent on PKA stimulation and ATP. CFTR single-channel measurements were performed as previously described (Aleksandrov and Riordan, 1998). Microsomal membrane vesicles were prepared from BHK-21 cells stably expressing wild-type CFTR. A planar lipid bilayer of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (Avanti Polar Lipids) at a 2:1 ratio was painted over a 0.2 mm aperture in a Teflon partition between cis and trans compartments of the chamber. Ion channels were transferred from CFTR-containing membrane vesicles into the preformed lipid bilayer as a result of spontaneous fusion. All measurements were taken at 30°C in symmetrical salt solution containing: 300 mM Tris-HCl pH 7.2, 3 mM MgCl₂ and 1 mM EGTA. Tris-HCl salt was used to exclude recording of native cation channels. Single-channel currents were measured at −75 mV under voltage-clamp conditions using an Axopatch 200B amplifier. (A) No CFTR ion channels can be recorded before addition of ATP and PKA to the cis compartment (recorded for 60 minutes, a representative record is shown). (B) 2 mM Na₂ATP and 100 units/ml PKA catalytic subunit were added to the cis compartment to activate CFTR. The opening of a CFTR Cl⁻ channel was monitored after a 10-minute incubation as a jump down to a more negative current. Therefore, in the single-channel record shown, the lower current level corresponds to the open state, whereas the upper level corresponds to the closed state.

• Supplemental Figure S2 -

  Fig. S2. Calreticulin does not interact with CFTR. CFTR was immunoprecipitated with mouse monoclonal anti-CFTR antibody 596 crosslinked to Dynabeads Protein G. CFTR was detected with anti-CFTR antibody 596, calnexin (CNX) with rabbit polyclonal antibody SPA860 (Stressgen) and calreticulin (CRT) with rabbit polyclonal antibody PA3-900 (Affinity Bioreagent). Cell lysates representing 2% of total inputs for immunoprecipitation are shown on the right.

• Supplemental Figure S3 -

  Fig. S3. Glycosidase inhibitors inhibit complex glycosylation of CFTR. BHK-21 cells were transiently transfected with CFTR and treated immediately after transfection with KIF (kifunensine, 0.2 mM), DMM (1-deoxymannojirimycin, 0.5 mM), CAS (castanospermine, 0.2 mM) or no drug as control. Twenty hours later, cell lysates were prepared and 35 μg of protein analyzed by western blotting using mouse monoclonal anti-CFTR antibody 596.

• Supplemental Figure S4 -

  Fig. S4. CFTR N900D is stabilized in the presence of ALLN. CHO-K1 cells stably expressing CFTR N894D, CFTR N900D or CFTR N894D/N900D were treated for 18 hours with BFA (10 μg/ml) or BFA and ALLN (50 μM). Cell lysates (25 μg) were subjected to SDS-PAGE and western blotting. CFTR was detected with mouse monoclonal anti-CFTR antibody 596. Actin was detected with an anti-actin polyclonal antibody (Lab Vision Corporation) as loading control.