The following antibodies were used in this study: mouse monoclonal anti-CFTR (C-terminus specific) (clone 24-1; Genzyme/Techne, Cambridge, MA), rabbit polyclonal anti-calnexin (C-terminus specific) (anti-CNX; SPA-860; Stressgen Biotechnologies, Victoria, BC, Canada), rabbit polyclonal anti-ubiquitin (anti-Ub; FL-76; Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-ubiquitinated proteins (anti-Ub-Protein; clone FK2; Affiniti Bioreagents, Golden, CO), mouse monoclonal anti-20S proteasome subunit α7 (anti-20S; clone HC8; Affiniti Bioreagents), mouse monoclonal anti-vimentin (clone V9; Santa Cruz Biotechnology), mouse monoclonal anti-Hsp70 (clone C92F3A-5; Stressgen Biotechnologies), rat monoclonal anti-Hsc70 (clone 1B5; SPA-815; Stressgen Biotechnologies), rabbit polyclonal anti-calreticulin (anti-CRT; SPA-600; Stressgen Biotechnologies), mouse monoclonal anti-KDEL (anti-BiP; clone 10C3; Stressgen Biotechnologies), rabbit polyclonal anti-protein disulfide isomerase (PDI) (SPA-890; Stressgen Biotechnologies), mouse monoclonal anti-β-COP (clone maD; Sigma-Aldrich, St. Louis, MO), mouse monoclonal anti-KDEL receptor (anti-KDELr; clone KR-10; Stressgen Biotechnologies), mouse monoclonal anti-Golgi 58K protein (anti-p58; clone 58K-9; Sigma-Aldrich), mouse monoclonal anti-Lamp-1 (clone UH1; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), and mouse monoclonal anti-FLAG M2 (Sigma-Aldrich) antibodies.
MG-132 and nocodazole were purchased from Calbiochem (San Diego, CA), and brefeldinA and cycloheximide were purchased from Sigma-Aldrich.
Baby Hamster kidney (BHK) cells stably expressing green fluorescent protein (GFP)-CFTR (CFTR-BHK) and GFP-ΔF508 CFTR (ΔF508-BHK), and Chinese hamster ovary (CHO) cells stably expressing CFTR (CFTR-CHO) and ΔF508 CFTR (ΔF508-CHO) were grown and maintained as described previously (Lukacs et al., 1994
; Loo et al., 1998
). CFTR- and ΔF508-CHO cells were maintained in α-minimal essential medium (MEM) containing 7% fetal bovine serum, antibiotics, and 200 μM methotrexate. CFTR- and ΔF508-BHK cells were maintained in DMEM/F-12 medium containing 10% fetal bovine serum, antibiotics, and 500 μM methotrexate. CFBE41o-
cells were grown in MEM with 10% fetal bovine serum and antibiotics on glass-bottomed culture dishes coated with human fibronectin. To increase CFTR expression, cells were incubated with 1 mM (CFTR- and ΔF508-BHK cells) or 2 mM sodium butyrate (CFTR-, ΔF508-CHO, 16HBE14o-
, and CFBE41o-
cells) for 20-24 h before analysis.
Recombinant adenovirus expressing human calnexin (Ad-CNX) or LacZ (Ad-LacZ) based on adenovirus 5 (Ad5) was produced as described previously (Okiyoneda et al., 2002
SDS-PAGE and Western Blotting
Subconfluent (90-100%) CFTR- and ΔF508-CHO cells grown on six-well plates were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed at 4°C in 100 μl of radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mg/ml sodium deoxycholate, and 1% NP-40) containing 1% protease inhibitor cocktail (Sigma-Aldrich) and centrifuged at 15,000 × g for 10 min at 4°C. The supernatant was prepared as cell lysates (detergent-soluble fraction). To prepare the detergent-insoluble fraction, the precipitate was washed three times with RIPA buffer, solubilized in 40 μl of 10 mM Tris-HCl, 1% SDS for 10 min at room temperature (RT), and 160 μl of RIPA buffer, supplemented with protease inhibitor cocktail, was added (final concentration 1%). Insoluble fractions were passed several times through a 27-gauge needle to decrease the viscosity. The detergent-soluble and -insoluble fractions were subjected to SDS-PAGE on 6 or 7.5% polyacrylamide gels. Protein was electroblotted from the gels to polyvinylidene difluoride membrane. The membranes were blocked in 5% skim milk and 0.1% Tween 20 in PBS for 1 h at RT, and incubation with the primary antibodies specified in the figure legends was for 1 h at RT. The membranes were then washed three times in 0.05% Tween 20 in PBS and incubated with the appropriate secondary antibody for 1 h at RT. After washing, membranes were incubated with ECL detection reagents (Amersham Biosciences, Piscataway, NJ) and analyzed by luminescent image analyzer (LAS-1000; Fujifilm, Tokyo, Japan).
Pulse-Chase Analysis and Immunoprecipitation
Pulse-chase analysis and immunoprecipitation were carried out as described previously (Lukacs et al., 1994
). Subconfluent CFTR- and ΔF508-CHO cells infected with or without Ad-CNX were incubated for 30 min in methionine- and cysteine-free αMEM 48 h after infection, and then pulse-labeled for 20 min with 100 μCi/ml [35
S]methionine and [35
S]cysteine (>1000 Ci/mmol; Amersham Biosciences). For chasing, the cells were washed three times with PBS and the labeling medium was replaced by complete αMEM containing 1 mM cycloheximide. Radiolabeled CFTR was isolated by immunoprecipitation. The cells were washed with ice-cold PBS three times and then lysed at 4°C in 1 ml of RIPA buffer containing 1% protease inhibitor cocktail. Samples were centrifuged at 15,000 × g
for 10 min at 4°C, and the supernatant was incubated for 2 h at 4°C with monoclonal anti-CFTR antibody immobilized in protein G Sepharose 4 Fast Flow (Amersham Biosciences). Immune complexes were precipitated, followed by four washes with 1 ml of RIPA buffer. Immunoprecipitated proteins were eluted for 15 min at 37°C with 2× concentrated loading buffer and analyzed on 6% SDS-PAGE gels. The gels were dried and analyzed with a BAS imaging plate scanner (BAS-2000; Fujifilm). The radioactivity associated with CFTR was quantified using Image Gauge software (version 3.4; Fujifilm).
Transfection was performed using TransIT-LT1 transfection reagents (Mirus, Madison, WI). Subconfluent cells grown on glass-bottomed culture dishes were transfected with 2 μg of plasmid DNA per dish.
Small Interfering RNA (siRNA) Preparation and Transfection
Specific siRNA was designed as described previously (Elbashir et al., 2001
). We used the 21-nucleotide (nt) sense strand (5′-GCAUCAUGCCAUCUCUGCUdTdT, coding region 384-402 relative to the start codon) and the 21-nt antisense strand (5′-AGCAGAGAUGGCAUGAUGCdTdT) of calnexin mRNA (GenBank accession no. AB071869). siRNA duplex was prepared as described previously (Elbashir et al., 2001
). Transient transfection with siRNA was performed by using Trans
IT-TKO (Mirus) as described by the manufacturer. siRNA duplex was used at a concentration of 10 nM.
Immunocytochemical Analysis and Confocal Laser Scanning Microscopy
Subconfluent cells grown on glass-bottomed culture dishes were washed twice with PBS, fixed in 3.7% paraformaldehyde in PBS for 20 min at RT, and permeabilized with 0.1% Triton-X in PBS for 20 min at RT. For immunostaining with anti-Golgi 58K protein or Lamp-1 antibodies, cells were permeabilized with 0.5% Triton-X in PBS or immunofluorescence buffer (150 mM NaCl, 5 mM NaN3
, 1 mM EDTA, 15 mM Tris-Cl, 0.1% gelatin, 0.01% saponin, pH 7.5) for 20 min at RT (Wimer-Mackin and Granger, 1996
). All cells were washed twice with PBS, subsequently incubated in PBS containing 1% bovine serum albumin (BSA) (BSA/PBS) for 30 min at RT, and then incubated for 1 h with corresponding primary antibodies (1:100 dilution) in 1% BSA/PBS at RT. Cells were washed three times with PBS and then immunostained with fluorescein isothiocyanateor tetramethylrhodamine B isothiocyanate (TRITC)-conjugated secondary antibodies (1:100 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA) or Alexa Fluor 488-conjugated secondary antibody (1:100 dilution; Molecular Probes, Eugene, OR.) in 1% BSA/PBS for 45 min at RT. Cells were washed three times with PBS and mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Cells were observed and analyzed with a Fluoview FV300 confocal laser scanning microscope (Olympus, Tokyo, Japan).
Fluorescence Recovery after Photobleaching (FRAP) Analysis
ΔF508-BHK cells infected with Ad-CNX were washed twice with PBS and the culture medium was replaced with CO2-independent medium (Invitrogen, Carlsbad, CA) containing 1 mM cycloheximide, either immediately or after fixation, in 3.7% paraformaldehyde in PBS for 20 min at RT. The temperature was maintained at 37°C. FRAP analysis was carried out on a FV300 confocal laser scanning microscope with a 60×, 1.25 numerical aperture objective (Olympus). A single z-section was imaged before and at 5- to 7.5-s intervals after photobleaching. The photobleaching was carried out at a wavelength of 488 nm at maximal power for iterations. The fluorescence intensities of the photobleached region were obtained using Fluoview software (version 3.3), and data were analyzed using Microsoft Excel.
Transmission Electron Microscopy
ΔF508-BHK cells infected with or without Ad-CNX were fixed in 1.0% glutaraldehyde for 30 min and postfixed in 1.0% osmium tetroxide for 30 min at 4°C. After dehydration in a graded ethanol series, the cells were embedded in epoxy resin. Ultrathin sections stained with uranyl acetate and lead citrate were observed with an H-7500 electron microscope (Hitachi, Tokyo, Japan).