S1
Supporting information
Supporting information: Methods
Materials.
The pcDNA3.1/Myc-His vector was purchased from Invitrogen Life
Technologies (Carlsbad, CA, USA).
The Adeno-X Tet-Off Expression System 1, containing
the pTRE-shuttle2 vector and adeno X viral DNA, was purchased from Takara Bio, Inc
(Shiga, Japan).
The Cholesterol Cell-Based Detection Assay Kit was purchased from
Cayman Chemicals (Ann Arbor, MI, USA).
All other chemicals used were commercially
available and of reagent grade.
Cell culture.
CHO-K1 cells were cultured in Ham’s F-12 medium (Wako, Osaka.,
Japan), and HEK293 cells and HepG2 cells were cultured in Dulbecco’s modified Eagle’s
medium [4.5 g/L glucose] (DMEM) (Nacalai Tesque, Inc., Kyoto, Japan). All media was
supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 g/ml) (GIBCO
BRL, Gaithersburg, MD, USA).
Construction of NPC2-Myc-His and GM2AP-Myc-His vectors.
Human NPC2
cDNA (accession number, BC002532) was purchased from the I.M.A.G.E consortium
(I.M.A.G.E clone no. 3140870).
Human GM2AP cDNA (accession number, NM000405)
S2
was amplified by PCR from total RNA of HepG2 cells. Subsequently, NPC2 and GM2AP
cDNAs were inserted into pcDNA3.1/Myc-His vector plasmids to express each cDNA with
an attached Myc tag sequence (EQKLISEQDL) and a 6×His tag sequence at the 3´-end.
The following sense and anti-sense primers were used to construct each mutant
NPC2-expressing
vector:
D72A
(215A>c)
5´-CATTCCTGAGCCTGcTGGTTGTAAGAGTGG-3´;
(sense
antisense
primer,
primer,
5´-CCACTCTTACAACCAgCAGGCTCAGGAATG-3´); N58Q (172A>c and 174T>g)
(sense primer, 5´-CAGTCTACAGCGTCcAgGTCACCTTCACCAGC-3´; antisense primer,
5´-GCTGGTGAAGGTGACcTgGACGCTGTAAGACTG-3´); and N135Q (403A>c and
405C>g)
(sense
primer,
5´-CTTCAGGATGACAAAcAgCAAAGTCTCTTCTGC-3´;
antisense primer, 5´-GCAGAAGAGACTTTGcTgTTTGTCATCCTGAAG-3´).
Immunoblot analysis.
Target cDNA was introduced into cultured cells grown to
approximately 70% confluence by transfection (for the expression vector) or infection (for
the adenoviruses).
Twenty-four hours after introducing each cDNA, the cells and the
corresponding culture media were harvested separately. Media from adenovirus-infected
cells were concentrated using Centricon YM-10 centrifuge filters (Millipore Corporation,
Billerica, MA, USA).
Cell pellets were lysed with Ripa buffer (0.1% SDS, 0.5%
S3
deoxycholate and 1% Nonidet P-40). The protein concentrations were determined by the
Lowry method (1) using bovine serum albumin (BSA) as a standard.
Total cell lysate and
concentrated media diluted with 2×SDS loading buffer were subjected to immunoblot
analysis as described previously (2, 3).
In order to separate each protein, 12.5%
SDS-polyacrylamide gels were used for the detection of NPC2, GM2AP, cathepsin D and
-tubulin and 7% gels were used for the detection of NPC1L1. Molecular weights were
determined using a prestained protein marker (New England BioLabs, Bevery, MA, USA).
The primary antibodies used for experiments were as follows: rabbit anti-His (BioVision ,
Mountain View, CA, USA) for the detection of the His tag (used at a 1:5000 dilution), mouse
anti-Myc (Roche Applied Science, Indianapolis, IN, USA) for the detection of the Myc tag
(1:1000), rabbit anti-HA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for the
detection of the HA tag (1:1000), rabbit anti-NPC1L1 (Cayman Chemicals, Ann Arbor, MI,
USA) (1:200), rabbit anti-NPC2 (Santa Cruz Biotechnology, Inc.) (1:200), goat anti-cathepsin
D (Santa Cruz Biotechnology, Inc.) (1:200), rabbit anti-NPC1 (GeneTex Inc, Irvine, CA,
USA) (1:500), and rabbit anti--tubulin (Cayman Chemicals) (1:1000).
For detection, the
membrane was incubated for 1 hour at room temperature with a 1:5000 dilution of
horseradish
peroxidase-labeled
secondary
antibody
[anti-mouse
(GE
Healthcare,
Buckinghamshire, UK), anti-rabbit (GE Healthcare) or anti-goat (Santa Cruz Biotechnology,
S4
Inc.) IgG] in TBS-T containing 0.1% BSA. Peroxidase activity was assessed using the ECL
Plus Western Detection System (GE Healthcare) with a luminescent image analyzer (Bio-Rad
Laboratories, Tokyo, Japan).
Co-immunoprecipitation
Cell lysates from CHO-K1 cells infected with the
indicated adenoviruses were rotated overnight at 4°C with 4 g of mouse anti-Myc antibody
or 4 g of goat anti-HA antibody (Novus Biologicals, Inc., Littleton, CO, USA).
Twenty
microliters of a 50% slurry of washed Bio-Adembeads Protein G (Ademtech, Pessac, France)
was added and the samples were rotated slowly at 4°C for an additional 3 hours. Beads
were collected magnetically, washed with Ripa buffer at least five times, and incubated at
37°C for 30 minutes with SDS loading buffer containing dithiothreitol.
The samples were
centrifuged briefly and the supernatants were analyzed by immunoblot analysis.
Quantitative real-time PCR.
To determine the mRNA levels of NPC2 and NPC1L1,
cells were harvested in RNA-Solv reagent (OMEGA bio-tek, Inc. Lilburn, GA, USA) and
prepared RNA was reverse-transcribed with ReverTra Ace (Toyobo, Osaka, Japan).
Quantitative real-time PCR was then performed, as described previously (4, 5). Primers
were used as follows: NPC2 sense sequence (5´-ACAGCGTCAATGTCACCTTC-3´), NPC2
S5
antisense sequence (5´-CATCCTGAAGTTGCCACTCCAC-3´), NPC1L1 sense sequence
(5´-GGTATCACTGGAAGCGAGTC-3´),
NPC1L1
-actin
(5´-AGGTAGAAGGTGGAGTCGAG-3´),
(5´-CCGGAAGGAAAACTGACAGC-3´)
and
-actin
antisense
sequence
sense
sequence
antisense
sequence
(5´-GTGGTGGTGAAGCTGTAGCC-3´).
Glycosidase digestions.
Twenty micrograms of protein from cellular extracts or 10
l of concentrated media prepared as described above were digested using Endo H or
PNGase F according to the manufacturer’s instructions (New England Biolabs).
Immunohistochemical staining.
HepG2 cells at 70% confluence cultured on 35 mm
glass dishes were transfected with the NPC1L1-HA expression vector.
After 24 hours, cells
were left untreated or were treated with MG132 (10 M) for 6 hours and were subsequently
washed with phosphate buffered saline (PBS) and fixed with Bouin’s fixative for 1 hour at
room temperature.
Proteins were detected with the following antibodies diluted as indicated
in PBS containing 0.1% BSA: rabbit anti NPC2 (1:200), mouse anti-HA (Santa Cruz
Biotechnology, Inc.) (1:100), goat anti-cathepsin D (1:100), or goat anti-calnexin (Santa Cruz
Biotechnology, Inc.) (1:100). Secondary antibodies (diluted 1:250 in PBS containing 0.1%
S6
BSA) were Alexa Fluor 488 donkey anti-rabbit IgG, Alexa Fluor 647 donkey anti-mouse IgG
and Alexa Fluor 546 donkey anti-goat IgG (Molecular Probes, Eugene, OR, USA) for the
immunodetection of NPC2, NPC1L1-HA, and cathepsin D and calnexin, respectively. Cells
were analyzed by confocal microscopy (Olympus, Tokyo, Japan).
Transfection of siNPC1L1 into HepG2 cells. RNAi studies were carried out as
reported previously (4, 5) in HepG2 cells in order to determine the effect of NPC1L1
knockdown on the expression of endogenous NPC2.
DMEM to a density of 2.0 × 105 cells/ml.
HepG2 cells were suspended in
Approximately 20 pmol/well of NPC1L1 siRNA
(5´-CCAGCUACAUUGUCAUAUUTT-3´) or a control siRNA designed not to interfere with
any human genes (Sigma Aldrich, Inc., St Louis, MO, USA) was added to each 35 mm dish
in a volume of 800 l of DMEM.
Twelve microliters of Lipofectamin RNAiMAX
(Invitrogen Life Technologies) was then added to each dish containing the diluted siRNA
molecule. After incubation for 20 minutes at room temperature, 4 ml of cell dilution was
added to each dish.
After 2 days of incubation, cells were harvested and the mRNA and
protein levels of NPC1L1 and NPC2 were determined by quantitative real-time PCR and
immunoblot analysis, respectively.
S7
Purification of recombinant NPC2.
In order to purify each NPC2-Myc-His protein
[wild-type (WT) and D72A mutant protein], Ad-NPC2-Myc-His and Ad-tTA were used to
infect HEK293 cells grown on 10 cm dishes. The next day, the culture media was harvested
and filtered through a 0.22 m syringe filter. Conditioned media samples were concentrated
using Centriprep YM-10 concentrators (Millipore Corporation, Billerica, MA, USA) and
bound to 0.1 g of IMAC Ni-charged resin (Bio-Rad Laboratories) for 2 hours at 4°C under
gently rotation. The mixture was then loaded onto an appropriately-sized column, which
was washed with five column volumes of washing buffer (50 mM sodium phosphate, 300
mM NaCl and 10 mM imidazole adjusted to pH 8.0).
Elution of each type of
NPC2-Myc-His protein was carried out using two column volumes of elution buffer (washing
buffer containing 500 mM imidazole adjusted to pH 8.0). The eluate was concentrated and
separated from imidazole by centrifugation with a Centricon YM10 centrifuge filter
(Millipore Corporation).
The samples were then precipitated with acetone in order to
remove lipids from NPC2-Myc-His proteins. The concentrations of the recombinant NPC2
proteins were determined by the Lowry method (1) using BSA as a standard.
S8
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