Methyl-β-cyclodextr in (MβCD) and anti-neutrophil myeloperoxidase [MPO; mouse anti-goat polyclonal IgG (H+L)] antibodies were obtained from Santa Cruz Biotechnology, Inc. (1:100, cat. no. sc-16129, Dallas, TX, USA), along with lipid-raft disruptor filipin. Alexa Fluor 488-cholera toxin subunit B (CTXB) and Alexa Fluor 594-labeled chicken anti-Goat IgG (H+L) secondary antibody were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Anti-flotillin-1 antibody was purchased from BD Biosciences (1:1000, cat no. 61802; Franklin Lakes, NJ, USA). Polyclonal anti-ERp29 rabbit anti-mouse antibody was obtained from Abcam (1:3000, ab11420; Cambridge, MA, USA). Optiprep was obtained from Axis-Shield, Inc. (Norton, MA, USA). High glucose Dulbecco's modified Eagle's medium (DMEM) was purchased from GE Healthcare Life Sciences (Logan, UT, USA). An Enhanced Chemiluminescence (ECL) kit was obtained from PerkinElmer Inc. (Waltham, MA, USA). Human LDL was purchased from ProSpec-Tany TechnoGene, Ltd. (Ness Ziona, Israel).

Raw264.7 mouse macrophages were purchased from the China Centre for Type Culture Collection (Wuhan, China). The cells were cultured in high glucose DMEM supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin (both from Thermo Fisher Scientific, Inc.) at 37°C and in 5% CO₂. When cell density reached 70–80% confluence, the cells were washed three times with phosphate-buffered saline and pre-incubated for 2 h in serum-free DMEM for LDL oxidation. The Raw264.7 cell line was incubated with native-LDL (100 µg/ml) at 37°C for 3, 6, 12 and 24 h.

To disrupt lipid raft membrane domains, membrane cholesterol was depleted by treating the Raw264.7 cells with DMEM supplemented with 5 mM MβCD for 30 min or 100 nM filipin for 15 min at 37°C.

The TBA assay was used to assess the extent of cell-mediated LDL oxidation as described previously (20). TBA reacts with malondialdehyde (MDA) and MDA-like derivatives to form TBA reactive species, which may be quantified by spectrophotometry at 535 nm using a UV-2000 spectophotometer [UNICO (Shanghai) Scientific Instrument Co., Ltd.]. Data are presented as MDA equivalents (nM MDA/mg protein).

Detection of lipid rafts was performed as described previously (21). Briefly, the Raw264.7 cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) and double stained with Alexa Fluor 488-CTXB (which attaches to ganglioside GM1 and thus does not require a second antibody), or incubated with anti-MPO antibody followed by Alexa Fluor-594-labeled secondary antibody, prior to being visualized under a DM6000 confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany).

Detergent-free rafts were prepared using the OptiPrep gradient method previously described by Macdonald and Pike (22). Following centrifugation at 52,000 × g for 90 min, cloudiness was observed throughout the gradient. A diffuse band was observed about one-third of the way down the gradient, and a distinct band was apparent at the interface of the 20% end of the gradient and the 25% OptiPrep bottom layer. The gradients were fractionated into 0.67 ml fractions (each in a new tube), and the distribution of various proteins was assessed by western blotting as described below.

Total protein from each fraction was determined using a bicinchoninic acid assay kit (Thermo Fisher Scientific, Inc.). The proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis prior to being electrophoretically transferred onto nitrocellulose membranes (GE Healthcare Life Sciences). The membranes were blocked using 5% non-fat milk in Tris-buffered saline with Tween 20 (TBST) for 1 h, incubated with primary antibodies against anti-flotillin-1, ERp29 and transferrin (1:2,000; cat. no. ab84036, Abcam) in TBST overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit/anti-mouse secondary antibodies [1:1,000; cat. nos. A0208, A0216; IgG (H+L); Beyotime Institute of Biotechnology, Haimen, China]. Prior to being washed three times with TBST. Protein bands were visualized on X-ray film (Kodak, Rochester, NY, USA) and quantified using Image J software (version 1.48U; National Institutes of Health, Bethesda, MA, USA).

In-gel digestion with trypsin was performed as previously described (23–25). Peptide samples were resuspended in loading buffer (containing 0.1% formic acid, 0.03% trifluoroacetic acid and 1% acetonitrile) and loaded onto a 15 cm nano-HPLC column (internal diameter 100 µm) packed with Reprosil-Pur 120 C18-AQ 1.9 µm beads (Dr Maisch HPLC GmbH, Ammerbuch, Germany), and eluted for 120 min with 4–80% buffer B reverse phase gradient (buffer A, 0.1% formic acid and 1% acetonitrile in water; buffer B, 0.1% formic acid in acetonitrile) generated by a NanoAcquity UPLC system (Waters Corporation, Milford, MA, USA). The peptides were ionized with 2.0 kV electrospray ionization voltage from a nano-ESI source (Thermo Fisher Scientific, Inc.) on a hybrid LTQ XL Orbitrap mass spectrometer (Thermo Fisher Scientific, Inc.).

Data-dependent acquisition of centroid MS spectra at 30,000 resolution and MS/MS spectra were obtained in the LTQ following collision-induced dissociation (collision energy, 35%; activation Q, 0.25; and activation time, 30 msec) for the top 10 precursor ions, with charge determined by the Sage Sorcerer SEQUEST (version 3.5; Sage-N Research, Inc., Milpitas, CA, USA) acquisition software to be z≥2. Dynamic exclusion of peaks already sequenced at 30 sec with early expiration for two count events with signal-to-noise >2. Automatic gating control was set to 150 msec maximum injection time. The Sage Sorcerer SEQUEST (version 3.5; Sage-N Research, Inc.) was used to search and match MS/MS spectra to a complete semi-tryptic mouse proteome database (NCBI reference sequence revision 54, with 34,421 target entries; http://www.ncbi.nlm.nih.gov/refseq/) and a pseudo-reversed decoys sequences (26,27) with a 20 ppm mass accuracy threshold. Static modifications for cysteine carbamidomethyl (+57.021465) and dynamic modifications for oxidized methionine (+15.99492) were included. Only b and y ions were considered for scoring (Xcorr), and Xcorr along with ΔCn were dynamically increased for groups of peptides organized by a combination of trypticity (fully or partial) and precursor ion charge state in order to remove false positive hits along with decoys until a false discovery rate (FDR) of <1% was achieved.

The FDR was estimated by the number of decoy matches (nd) and total number of assigned matches (nt): FDR=2nd/nt, assuming mismatches in the original database were the same as in the decoy database (28). Quantification of proteins was based on the comparison of extracted ion current intensities for identified peptides as previously described (29).

The upregulated proteins were analyzed using the Panther classification system (http://www.pantherdb.org/) and were assigned to various functional groups.

All values are presented as the mean ± standard deviation. Statistical analyses were performed using GraphPad Prism software (version 5; GraphPad Software, Inc., La Jolla, CA, USA). The paired, two-tailed Student's t-test was used to analyze the significance of the difference between groups. P<0.05 was considered to indicate a statistically significant difference.

Previous studies have demonstrated that ox-LDL induced aggregation of gp^91phox (30) and Fas (31) in lipid rafts. In order to examine whether native-LDL induces lipid raft clustering, GM1 ganglioside (the predominant lipid raft component) was stained using Alexa-488-labeled CTXB. As shown in Fig. 1, native-LDL stimulation caused an aggregation of GM1, exhibited as green dots or patches. These results suggest that treatment with native-LDL led to the formation of lipid rafts in Raw264.7 cells.

To examine whether MPO was able to aggregate in lipid raft clusters following native-LDL stimulation, Raw264.7 cells were stained with Alexa-488 CTXB and Alexa-594-conjugated anti-MPO, and the distribution of MPO in lipid raft clusters was visualized by confocal microscopy. As shown in Fig. 2, MPO, an important enzyme involved in the oxidation of LDL, was distributed in both the membrane and cytosol of cells. After LDL treatment, MPO was redistributed in the plasma membrane and colocalized with GM1, so LDL increased MPO translocation into lipid rafts. This colocalization was blocked in MβCD-treated cells.

The role of lipid rafts in macrophage-mediated LDL oxidation was investigated. Fig. 3 shows that Raw264.7 cells oxidized human LDL over a 24 h time period, which was significantly decreased following treatment with MβCD and filipin compared with the controls.

Lipid rafts were isolated and purified from non-treated and LDL-stimulated Raw264.7 cells using OptiPrep gradient centrifugation. As shown in Fig. 4, low protein concentration levels were present in the lipid raft fractions (fraction 6–8). The highest protein concentration levels were located at the bottom of the gradient. However, an increase in total protein concentration levels was observed in fraction 7, and the total protein levels in the lipid rafts fractions significantly increased following treatment with LDL (P<0.05).

To identify differentially regulated lipid raft-associated proteins following LDL stimulation, label-free quantitative proteomics analysis was performed on lipid raft fractions of macrophages. The distribution of the ratios of protein abundance between the LDL-stimulated macrophages and the resting state is shown in Fig. 5A. The present study identified 1,449 proteins with ≥1 unique peptides with quantifiable abundance measurements, of which many were lipid raft marker proteins, including flotillins and glycosylphosphatidylinositol-anchored proteins. From the identified proteins, a fold change of ≥2 was used to define differential regulation. Out of the 1,449 proteins, 204 and 203 proteins were shown to be downregulated and upregulated following LDL stimulation, respectively. Protein groups were then sorted according to biological processes, cellular components and molecular function GO categories (Fig. 5B). Upregulated proteins comprised GO terms associated with metabolic processes and response to stimuli. Notably, the results also demonstrated enrichment for biological adhesion, localization, and enzyme regulator activity. Furthermore, apoB100 was identified in lipid rafts when the protein spectra were matched to a human proteome database, which indicated the human origin of LDL.

The 203 upregulated proteins were analyzed using STRING. Numerous interaction groups were apparent, such as Hmox-1 - Bax, Pfn - Cap-1. Notably, ERp29 was associated with calreticulin.

To further validate the proteomic identification results, the expression levels of one of the upregulated proteins, ERp29, were analyzed by western blotting. The floated low density fractions 6, 7 and 8 (lipid rafts observed one-third of the way down the gradient) represented the lipid raft fractions following gradient ultra-centrifugation of the cell lysates. As shown in Fig. 6A, the proteins from various fractions were immunoblotted for flotillin-1, a well-documented marker protein of lipid rafts using an anti-flotillin-1 antibody. It was demonstrated that flotillin-1 localized not only in lipid raft fractions, but also in non-raft fractions (high density fractions). It should be noted that this was not due to unsuccessful isolation of the lipid raft, but a phenomenon observed in previous studies (22,32). LDL induced a significant increase of the flotillin-1 into lipid raft, however, pre-treatment with MβCD almost entirely disrupted the lipid raft fractions, as determined by the lack of flotillin-1 in raft fractions. As shown in Fig. 6B, the expression levels of ERp29 in lipid rafts were significantly increased following treatment with LDL. This increase in expression levels was suppressed by MβCD pre-treatment. Notably, the transferrin receptor, a marker for the non-raft plasma membrane, was distributed at the bottom of the gradient (Fig. 6C), indicating that non-raft plasma membrane did not significantly contaminate the major lipid raft fractions. Bar graphs show the band density ratio of Flot-1 (Fig. 6D) and ERp29 (Fig. 6E), respectively.