The mouse hybridoma that produces mAb to the 9E10 c-Myc epitope (EQKLISEED) (Evan et al., 1985
) was purchased from the American Type Culture Collection (Rockville, MD). Rabbit polyclonal antibodies to transferrin were obtained from Dako A/S (Glostrup, Denmark). Antiserum 4, which recognizes the endogenous TGN38 protein of COS-7 cells (Wilde et al., 1992
), was a generous gift from Dr. G. Banting (University of Bristol, Bristol, United Kingdom). Peroxidase-conjugated rabbit anti-mouse immunoglobulin G (IgG) antibodies, peroxidase-coupled streptavidin, and sulfo-N
-hydroxyl-succinimido-biotin (sulfo-NHS-biotin) were from Pierce (Rockford, IL). Fluorescein- and Texas Red–conjugated secondary antibodies were from Southern Biotech (Birmingham, AL). Anti-FLAG M2 antibodies, Triton X-100, octyl-glucoside, neuraminidase, nocodazole, brefeldin A (BFA), chloroquine, monensin, NH4
Cl, and iron-saturated transferrin were obtained from Sigma (St. Louis, MO).
Cell Culture Conditions, Transfections, and DNA Constructs
Epithelial MDCK cells and COS-7 cells were grown on Petri dishes or glass coverslips in DMEM supplemented with 10% FBS (Life Technologies-BRL, Gaithersburg, MD), penicillin (50 U/ml), and streptomycin (50 μg/ml) at 37°C in an atmosphere of 5% CO2/95% air. Transfections were done by electroporation with the ECM 600 electroporation instrument (BTX, San Diego, CA). Selection of stable MDCK cell transfectants was carried out by treatment with 0.5 mg/ml G418 sulfate (Life Technologies-BRL) for 3 wk after transfection. Drug-resistant clones were picked up with cloning rings, and individual clones were screened for expression of MAL-FLAG by immunofluorescence analysis.
The MAL-FLAG construct, encoding MAL modified at its last extracellular loop by insertion of the sequence DYKDDDDK, which contains the FLAG epitope (DYKD), was generated by PCR with the use of the overlap extension technique (Ho et al., 1989
) and appropriate oligonucleotide primers. We have previously described the generation of an N
-glycosylated MAL protein, named MAL-Z, by insertion at the last extracellular loop of MAL of the 27-amino acid sequence (DYKDDDDKGNLSANITPYPYDVPDYAS) containing two tandem consensus N
-glycosylation sites (NLS and NIT) flanked by the FLAG and HA (YPYDVPYAS) epitopes and additional amino acids used as spacers (Puertollano and Alonso, 1999
). MAL-T was generated by substituting the asparagine residue within the two N
-glycosylation consensus sites that are present in MAL-Z with threonine with the use of the overlap extension technique (Ho et al., 1989
) and appropriate oligonucleotide primers. After cloning of the modified MAL cDNAs into the pCR3 eukaryotic expression vector (Invitrogen, Carlsbad, CA), the sequence of the inserted DNA was verified to check for possible amplification errors.
Detergent Extraction Procedures
GEMs were isolated by standard procedures (Brown and Rose, 1992
). Cells grown to confluence in 100-mm dishes were rinsed with PBS and lysed for 20 min in 1 ml of 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 at 4°C. The lysate was scraped from the dishes with a rubber policeman, the dishes were rinsed with 1 ml of the same buffer at 4°C, and the lysate was homogenized by passing the sample through a 22-gauge needle. The extract was finally brought to 40% sucrose in a final volume of 4 ml and placed at the bottom of an 8-ml 5–30% linear sucrose gradient. Gradients were centrifuged for 18 h at 39,000 rpm at 4°C in a Beckman (Fullerton, CA) SW41 rotor. Fractions of 1 ml were harvested from the bottom of the tube, and aliquots were subjected to immunoblot analysis.
After repeated washings with ice-cold PBS containing 0.1 mM CaCl2 and 1 mM MgCl2, cells were incubated with 0.5 mg/ml sulfo-NHS-biotin. After 30 min at 4°C, the solution was removed and the remaining unreacted biotin was quenched by incubation with ice-cold serum-free culture medium. Cell monolayers were washed with PBS and extracted with 0.5 ml of 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 60 mM octyl-glucoside for 30 min on ice. Extracts were then immunoprecipitated with mAb 9E10, and the immunoprecipitates were, or were not, digested with neuraminidase (0.05 U/ml) for 1 h at 4°C. Samples were finally fractionated by SDS-PAGE and analyzed with peroxidase-coupled streptavidin to trace the biotin groups incorporated during the surface-labeling procedure. Quantitative analyses were carried out with a computing densitometer.
Immunoblot and Immunoprecipitation Analyses
For immunoblot analysis, samples were subjected to SDS-PAGE in 15% acrylamide gels under reducing conditions and transferred to Immobilon-P membranes (Millipore, Bedford, MA). After blocking with 5% (wt/vol) nonfat dry milk, 0.05% (vol/vol) Tween-20 in PBS, blots were incubated with the indicated primary antibody. After several washings, blots were incubated for 1 h with goat anti-mouse IgG antibodies coupled to HRP, washed extensively, and developed using an ECL Western blotting kit (Amersham, Arlington Heights, IL).
For use in immunoprecipitation studies, antibodies were prebound overnight at 4°C to protein G–Sepharose in 10 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1% Triton X-100. Postnuclear supernatants prepared with 1% Triton X-100 plus 60 mM octyl-glucoside were incubated for 4 h at 4°C with a control anti-CD4 mAb bound to protein G–Sepharose. After centrifugation, the supernatant was immunoprecipitated by incubation for 4 h at 4°C with mAb 9E10 bound to protein G–Sepharose. The immunoprecipitates were collected, washed six times with 1 ml of 10 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1% Triton X-100, and analyzed by SDS-PAGE under reducing conditions.
Endocytosis Assays by Immunofluorescence Analysis or Neuraminidase Treatment
To detect the presence of MAL-FLAG on the cell surface or to monitor its internalization by immunofluorescence analysis, intact cells were washed several times with ice-cold PBS and incubated for 1 h at 4°C with anti-FLAG antibodies. After extensive washing with PBS to remove excess antibody, cells were incubated in normal medium at either 4 or 37°C to prevent or allow endocytosis, respectively. The cells were then fixed and permeabilized (surface plus internalized MAL-FLAG) or not (surface MAL-FLAG) with 0.2% Triton X-100 before the addition of the secondary fluorescent antibody. Samples were processed as described under Immunofluorescence Microscopy.
To monitor MAL-T internalization with the use of neuraminidase digestion, the surfaces of transfected cells were first subjected to biotinylation and then the cells were incubated at either 4 or 37°C for the indicated times to prevent or allow internalization, respectively. After washing extensively with PBS at 4°C, intact cells were rinsed twice with 25 mM 2-(N-morpholino)ethanesulfonic acid, pH 5.5, 2 mM CaCl2, 140 mM NaCl and incubated with 0.05 U/ml neuraminidase at 4°C for 1 h. After lysis, the extracts were subjected to immunoprecipitation with mAb 9E10, and the immunoprecipitates were subjected to immunoblot analysis with streptavidin conjugated to peroxidase and developed using the ECL Western blotting kit.
Flow Cytometric Internalization Assay
MDCK cells stably expressing MAL-FLAG were incubated for 60 min in the presence of anti-FLAG M2 mAb at 4°C, washed, and incubated for 15 min at either 4 or 37°C. Cells were then fixed with 3% paraformaldehyde and incubated with a fluorescein-conjugated secondary antibody to label the FLAG antibodies bound to MAL-FLAG that remained at the surface. Finally, samples were subjected to flow cytometric analysis using the Becton-Dickinson (Mountain View, CA) FACScan cytofluorimeter. To calculate the extent of internalization, the mean fluorescence intensity of each sample was calculated. The value obtained with background staining (anti-CD4 OKT4 mAb) was subtracted from that of each sample. The value corresponding to that of the cells incubated for 15 min at 37°C was divided by that of the cells incubated at 4°C to obtain the percentage of molecules remaining at the cell surface.
Treatments with 20 μM nocodazole or 5 μg/ml BFA were carried out in COS-7 cells incubated at 37°C for 30 min to allow internalization of anti-FLAG antibodies previously bound to surface MAL-FLAG for 1 h at 4°C. In the case of treatments with inhibitors of endosomal transport, 100 μM chloroquine, 10 μM monensin, or 50 mM NH4Cl was added to the culture medium either 30 min before or immediately before incubation at 37°C. In both cases, the drugs were maintained in the medium during the 37°C incubation period.
To analyze the total steady-state distribution of MAL, transfected cells grown on coverslips were washed twice with PBS, fixed in 3% paraformaldehyde for 15 min, rinsed, and treated with 10 mM glycine for 10 min to quench the aldehyde groups. The cells were then permeabilized with 0.2% Triton X-100, rinsed, and incubated with 3% BSA in PBS for 20 min. Coverslips were then incubated for 1 h with the primary antibody, rinsed several times, and incubated for 1 h with the appropriate fluorescent secondary antibody. For double-label immunofluorescence analysis, the procedure was repeated with the second primary and secondary antibodies. After extensive washing, the coverslips were mounted on slides. The cells were photographed with a Zeiss (Thornwood, NY) Axioskop photomicroscope using Kodak (Rochester, NY) T-Max 400 film. Primary antibodies included mouse 9E10 (IgG1) and anti-FLAG M2 (IgG1) mAbs and rabbit polyclonal antibodies to transferrin or to TGN38. Secondary antibodies included Texas Red–conjugated goat anti-mouse Igγ1 chain and fluorescein-conjugated anti-rabbit IgG antibodies absorbed against mouse IgG. Controls to assess the specificity and the lack of cross-labeling included incubations with control primary antibodies and omission of either of the primary antibodies.