QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online June 28, 2004
doi: 10.1242/10.1242/jcs.01182

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rhainds, D. Articles by Brissette, L. Search for Related Content PubMed PubMed Citation Articles by Rhainds, D. Articles by Brissette, L.

Localization and regulation of SR-BI in membrane rafts of HepG2 cells

Département des Sciences Biologiques, Université du Québec à Montréal, 1200 Saint-Alexandre, Montréal, Québec, H3B 3H5, Canada

View larger version (62K): [in a new window]   Fig. 1. Immunoblotting of HepG2 cell proteins fractionated by discontinuous sucrose gradient. HepG2 cell proteins were fractionated with detergent-free sodium carbonate discontinuous 5-40% sucrose gradients as described in the Materials and Methods. The 12 gradient fractions were concentrated with TCA and 50 µl sample of each fraction was loaded on 10% reducing SDS-polyacrylamide gel. After transfer on nitrocellulose, proteins were immunoblotted and detected by enhanced chemiluminescence. (A) SR-BI of control cells. (B) SR-BI of cells treated with 0.5% saponin (30 minutes at 4°C). C-F are from control cells: (C) caveolin-1; (D) clathrin heavy chain; (E) ABCA1; (F) L-FABP; (G) cytosolic NCEH; H, BSDL/CEL. The images are representative of three independent experiments.

View larger version (30K): [in a new window]   Fig. 2. Immunoblotting of Y1-BS1 cell proteins fractionated by discontinuous sucrose gradient. Y1-BS1 cell proteins were fractionated and immunoblotted as described in Fig. 1. (A) SR-BI; (B) caveolin-1 in Y1-BS1 cells. The images are one of two independent experiments.

View larger version (34K): [in a new window]   Fig. 3. Effect of COase and/or SMase treatment on [3H]CE and 125I-protein association of LDL and HDL3 in HepG2 cells. HepG2 cells were treated with 1 U/ml COase or 0.5 U/ml SMase or both enzymes for 1 hour at 37°C. The cells were then processed for [3H]CE association (black bars), CE selective uptake (CE association – protein association and degradation) (white bars), 125I-protein association (hatched bars) and degradation (cross-hatched bars), with 20 µg/ml radiolabelled LDL (A) or HDL for 3 hours at 37°C (B). Results are shown as mean percentage±s.e.m. Control values in µg protein/mg cell protein were set as 100% and were: 0.507±0.066, 0.444±0.066, 0.123±0.015, 0.029±0.001 for [3H]CE-LDL association (n=7), LDL-CE selective uptake, 125I-LDL protein association (n=7) and degradation (n=2) respectively; and 0.332±0.058, 0.271±0.058, 0.067±0.008 for [3H]CE-HDL3 association (n=7), HDL3-CE selective uptake and 125I-HDL3 protein association (n=7) respectively (mean±s.e.m.). Statistically different values from the control values (without enzymes) are indicated as: aP<0.05; bP<0.01 and cP<0.001.

View larger version (34K): [in a new window]   Fig. 4. Effect of filipin III or ß-CD treatment on [3H]CE and 125I-protein association of LDL and HDL3 in HepG2 cells. HepG2 cells were treated with 5 µg/ml filipin III or 10 mM ß-cyclodextrin for 1 hour at 37°C. The cells were then processed as described in Fig. 3. [3H]CE association (black bars), CE selective uptake (CE association – protein association and degradation) (white bars), 125I-protein association (hatched bars) and degradation (cross-hatched bars). Results are shown as mean percentage±s.e.m. Control values in µg protein/mg cell protein were set as 100% and were: 0.467±0.029, 0.311±0.025, 0.159±0.009, 0.041±0.004 for [3H]CE-LDL association (n=6), LDL-CE selective uptake, 125I-LDL protein association (n=6) and degradation (n=6) respectively; and 0.233±0.021, 0.159±0.023, 0.074±0.008, 0.002±0.004 for [3H]CE-HDL3 association (n=7), HDL3-CE selective uptake, 125I-HDL3 protein association (n=7) and degradation (n=7) respectively (mean±s.e.m.). Statistically different values from the control values (without treatment) are indicated as: aP<0.05; bP<0.01 and cP<0.001.

View larger version (34K): [in a new window]   Fig. 5. Effect of NEM treatment on [3H]CE and 125I-protein association of LDL and HDL3 in HepG2 cells. HepG2 cells were treated with 1 mM NEM for 30 minutes at 37°C. The cells were then processed as described in Fig. 3. [3H]CE association (black bars), CE selective uptake (CE association – protein association and degradation) (white bars), 125I-protein association (hatched bars) and degradation (cross-hatched bars). Results are shown as mean percentage±s.e.m. Control values in µg protein/mg cell protein were set as 100% and were: 0.607±0.076, 0.470±0.083, 0.136±0.012, 0.0301±0.0003 for [3H]CE-LDL association (n=9), LDL-CE selective uptake, 125I-LDL protein association (n=9) and degradation (n=6) respectively; and 0.313±0.029, 0.243±0.085, 0.070±0.003, 0.0020±0.0003 for [3H]CE-HDL3 association (n=9), HDL3-CE selective uptake, 125I-HDL3 protein association (n=9) and degradation (n=6) respectively (mean±s.e.m.). Statistically different values from the control values (without NEM) are indicated as: aP<0.05; bP<0.01 and cP<0.001.

View larger version (22K): [in a new window]   Fig. 6. Human SR-BI overexpression in HepG2 cells. HepG2 cells were stably transfected with a eukaryotic expression vector containing human SR-BI cDNA and clones were isolated. (A) SR-BI levels of normal HepG2 cells and of two overexpressing clones (S1.1 and S1.7) determined by immunoblotting with anti-SR-BI polyclonal antibody (1:5000) as described under Materials and Methods. Shown here is a representative image from five different protein extractions. (B) [3H]CE association (black bars) and 125I-protein association (white bars) of LDL (n=9) and HDL3 (n=9) in HepG2 cells overexpressing SR-BI measured as described in figure 3. (C) 125I-M-BSA association (black bars) and recycling (retroendocytosis, white bars) (n=4) in HepG2 cells overexpressing SR-BI measured by incubating 125I-M-BSA with cells at 20 µg/ml for 2 hours at 37°C according to the pulse-chase protocol described in Materials and Methods. Statistically different values from the control HepG2 cell values (or otherwise indicated) are indicated as: aP<0.05; bP<0.01 and cP<0.001.

View larger version (38K): [in a new window]   Fig. 7. Immunoblotting of lipid binding and/or sensor proteins in HepG2 cells overexpressing SR-BI. Total proteins were extracted by 1% Triton X-100 and were loaded on 10% reducing SDS-PAGE. 150 µg of protein were loaded for the caveolin-1 immunoblot (A), while 50 µg were loaded for L-FABP (B), cytosolic NCEH (C) and SREBP-2 (D) immunoblots. Shown here are representative images of at least four independent protein extractions.