#29.3
EXPERIMENTAL PROCEDURES
Cell Culture, Transfections, and Adenoviral Infection
HEK 293T cells were from the ATCC. HEK 293T-Reelin cells were a gift from Dr. Thomas Curran (Children's Hospital of Philadelphia). SNB-19 glioblastoma cells were a gift from Dr. Rene Bernards (The Netherlands Cancer Institute, Amsterdam, The Netherlands). The generation and maintenance of 3T3 mouse fibroblasts that stably produce VLDLR and Dab1 were previously reported (22). The cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum at 37 °C and 5% CO2. To collect Reelin-containing conditioned medium, HEK 293T-Reelin cells were grown to ∼75% confluence, washed twice with phosphate-buffered saline and cultured in Optimem medium (Invitrogen) for an additional 24 h. Conditioned medium was collected, filtered, and stored at −80 °C. HEK 293T cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. In experiments testing the ability of Idol to degrade other potential protein targets, a ratio of 3:1 (IDOL:target) was used. The generation of adenoviruses encoding Idol, GFP, and Sult2b1 was previously described (13, 23). SNB-19 cells were seeded (0.5 × 10624). cells/60-mm well) and infected with adenovirus the following day at a multiplicity of infection of 80. Primary hippocampal neurons were prepared from embryonic 17-day-old rats and cultured as described (24)
Plasmids and Expression Constructs
Expression plasmids for human IDOL, mouse Idol, HA-ubiquitin, LDLR, and LpR were previously reported (13, 25). The C-terminally tagged VLDLR-HA, VLDLR-GFP, and ApoER2-HA expression constructs were gifts from Dr. George Rebeck (Georgetown University). The FLAG-Lrp1b expression plasmid was a gift from Dr. Masashi Kawaichi (Nara Institute of Science and Technology, Japan). Full-length Drosophila melanogaster Dnr1 was cloned into the gateway plasmid pDONR221 (Invitrogen). To generate mammalian expression constructs for Dnr1, we used LR recombination between pDONR221-Dnr1 and an N-terminally V5 tag DEST plasmid (Invitrogen). Site-directed mutagenesis was used to introduce mutations in VLDLR-HA and V5-Dnr1 with the QuikChange multi-site mutagenesis kit (Stratagene). An amyloid precursor protein (APP) chimeric construct, N′-APP(1–675)-LDLR(780–860)-C′, that contains the APP ecto-domain and the transmembrane and intracellular domains of the LDLR fused to a C-terminal GFP was generated by standard cloning procedures. Restriction digest analysis and DNA sequencing were used to verify the correctness of all of the constructs used in this study.
Antibodies, Immunoblot Analysis, and Immunoprecipitation
Total cell or tissue lysates were prepared in radioimmune precipitation assay buffer (150 mm NaCl, 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% SDS, 100 mm Tris-HCl, pH 7.4) supplemented with protease inhibitors (Roche Applied Science). The lysates were cleared by centrifugation at 4 °C for 10 min at 10,000 × g. Protein concentration was determined using the Bradford assay (Bio-Rad) with bovine serum albumin as reference. The samples (10–40 μg) were separated on NuPAGE Bis-Tris gels (Invitrogen) and transferred to nitrocellulose. The membranes were probed with the following antibodies: LDLR (Cayman Chemical, 1:2000), tubulin (Calbiochem, 1:10000), GFP (affinity-purified rabbit polyclonal anti-GFP was a gift from Dr. Mireille Riedinger, 1:5000), LpR (2189/90, 1:500) (25), HA (Covance, 1:20000), V5 (Invitrogen, 1:5000), VLDLR (Santa Cruz clone 6A6, 1:250), α74 (1:20,000) (22), Reelin (Millipore clone G10, 1:200), Dab1 (D4 mouse monoclonal antibody was a gift from Dr. Andre Goffinet, 1:1000), and phosphotyrosine (Santa Cruz PY99, 1:200). Idol was detected with polyclonal antibodies raised in rabbits against Idol (13) or with a monoclonal antibody (Abcam, 1:500). Secondary horseradish peroxidase-conjugated antibodies (Zymed Laboratories Inc.) were used and visualized with chemiluminescence (Pierce). To immunoprecipitate HA-tagged proteins, equal amounts of protein of cleared lysates were incubated with EZ view red anti-HA affinity beads (Sigma) for 16 h. Subsequently, the beads were washed four times with radioimmune precipitation assay buffer. All of the incubations and washes were done at 4 °C with rotation. The proteins were eluted from the beads by boiling in 1× protein sample buffer for 5 min. The blots were quantified by densitometry.
RNA Isolation and Quantitative PCR
Total RNA was isolated from cells and mouse tissues using TRIzol (Invitrogen). One microgram of total RNA was reverse transcribed with random hexamers using iScript reverse transcription reagents kit (Bio-Rad). Sybergreen (Applied Biosystems) real time quantitative PCR assays were performed on an Applied Biosystems 7500HT sequence detector. The results show the averages of duplicate experiments normalized to 36B4. Primer sequences are available upon request.
Metabolic Labeling of Cells
HEK 293T cells were transfected with VLDLR-HA and Idol expression plasmids. Subsequently, the cells were washed twice with phosphate-buffered saline and pulsed for 15 min with Dulbecco's modified Eagle's medium lacking methionine and cysteine (Sigma) supplemented with 200 μCi/well easy Tag express 35S protein labeling mix (PerkinElmer Life Sciences). The cells were then washed three times and chased in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 μg/ml methionine, and 500 μg/ml cysteine for the indicated times. Preparation of cell lysates and immunoprecipitation of VLDLR-HA were conducted as detailed above.
Reelin Binding and Dab1 Phosphorylation Assays
Reelin binding assays were conducted essentially as described (22). Briefly, SNB19 cells were plated at a density of 0.5 × 106 cells/60-mm well. Subsequently, the cells were washed three times with Optimem medium (Invitrogen) and incubated with Reelin-containing conditioned medium on ice. Binding was allowed to proceed for 30 min, after which cells were vigorously washed five times with phosphate-buffered saline. Preparation of cell lysates and immunodetection was conducted as detailed above. Analysis of Dab1 phosphorylation following Reelin binding was done as described (22).
Animal Experiments
C57BL/6 mice (Jackson Laboratory) were fed a standard chow diet and housed in a temperature-controlled room under a 12-h light-dark cycle under pathogen-free conditions. The mice were orally gavaged with 20 mg/kg of the indicated LXR ligand. At the time of sacrifice, the tissues were collected, immediately frozen in liquid nitrogen, and stored at −80 °C. The tissues were processed for isolation of RNA and protein as above. The animal experiments were conducted in accordance with institutional guidelines.
Statistical Analysis
Real time PCR data and densitometry are expressed as the means ± S.D. Statistical analysis was done with a two-tailed Student's t test. A probability value of p < 0.05 was considered statistically significant.