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The protein levels in cells were examined using Western blot analysis as described for immunoblotting for LC3. To detect cytochrome c release, the mitochondria-free cytosolic protein fraction was isolated using a mitochondria isolation kit obtained from Thermo Fisher Scientific (Rockford, IL, USA). Antibodies against cytochrome c, mTOR, phospho-mTOR (Ser2481), phospho-mTOR (Ser2448), PARP, Bcl-xL, Bad and Bax were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against Bcl-2 and β-actin were obtained from Sigma. The intensity of the immunoreactive bands was determined using GeneTools software (Syngene, Frederick, MD, USA) after scanning the developed films. The results are expressed as the means ± standard deviation (SD) of three independent experiments.

The protein was isolated using cell lysis buffer (Cell Signaling, Beverly, MA, USA) and the concentrations were measured using the Bradford Protein Assay Kit (AMRESCO, Radnor, PA, USA). Protein lysates were prepared and Western blot analyses were performed as described previously [48 (link)]. For ubiquitination assay, cells were washed three times and then lysed with lysis buffer for 30 min. The cell lysate was centrifuged for 10 min at 12,000 rpm and the rest of homogenates were incubated with ubiquitin antibodies overnight at 4 °C. Protein A/G plus agarose beads (Santa Cruz) were added for another 2 h. The immunoprecipitation beads were washed with cold PBS five times, followed by Western blotting analysis. The levels of expression of β-actin and GAPDH were used as a gel loading control. Densitometric analyses of scanned immunoblotting images were performed using GeneTools software (Syngene version 6.05, Cambridge, UK). Results shown are representative of three separate experiments.

Complex formation of vaspin and KLK7 was performed as previously described [20 (link)]. Vaspin was incubated with 3 µM recombinant KLK7 (molar ratio protease/serpin 3:1) in the presence or absence of PtdInsPs (molar ratio PtdInsPs/vaspin 0.1, 1 or 10), ufh (molar ratio ufh/vaspin 10) or polyP (polyP₃ or polyP₄₅ 1−200 molar excess) for 1 min.
Proteins were separated using SDS-PAGE and gels were stained using Coomassie Brilliant Blue. Band intensities were determined by densitometric quantification (Gene tools software, Syngene) and were normalized to the reaction without PtdIns, ufh or polyP. Stoichiometry of inhibition (SI) was estimated from band intensities as previously described [20 (link)].

BMDMs were infected with L. monocytogenes as described above and lysed in lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 5 mM NaF, 2 mM sodium orthovanadate, 1 mM PMSF, 1X complete protease inhibitors). The whole cell lysates were mixed with Laemmli buffer (50 mm Tris–Cl, pH 6.8, 10% glycerol, 2% SDS, 0.1% bromophenol blue, 5% 2-mercaptoethanol), boiled for 5 min, separated on 4–20% polyacrylamide gels (BioRad), transferred to nitrocellulose membranes (Bio-Rad), blocked, and probed with the following primary antibodies: monoclonal Ab to iNOS (clone D6B6S, Cell Signaling) and polyclonal Ab to β-actin (Sigma). Membranes were developed using an ECL plus chemiluminescence kit (Thermo Fisher Scientific) and protein quantification on the Western blots was performed using GeneTools software (Syngene).

Insulin resistance was induced in HepG2 cells as previously described. Cells were washed with ice-cold PBS and lysed with a RIPA lysis buffer. For Western blotting, protein samples (20 μg) of sodium oleate induced insulin-resistant HepG2 cells were separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to a PVDF membrane and incubated with primary antibody (anti-NF-κB, anti-IRS1, anti-GLUT4, or anti-GAPDH), followed by a secondary antibody (horseradish peroxidase-conjugated anti-rabbit IgG). The intensity of the immunoblot signal was assayed using Western Bright ECL spray and analyzed quantitatively using GeneTools software from Syngene (Syngene, Cambridge, UK).
To choose the most effective concentration of PDTC, the cells were incubated for 26 h in serum-free DMEM at the different dosages of PDTC or 0.2% BSA and 200 μmol/L sodium oleate. The protein samples were prepared for Western blot experiments and incubated with anti-NF-κB.
Then, the cells were incubated for 2 h in serum-free DMEM and PDTC with the most effective concentration (all PDTC groups). After that, 0.2% BSA and 200 μmol/L sodium oleate and three dosages of OA were added. Next, the cells were incubated for an additional 24 h after preparing the protein samples in Western blot experiments and incubated with anti-IRS1.

DNA extraction from fecal pellets and pyrosequencing analyses were performed as previously described [10 (link),57 (link)]. Composition of the microbiota was assessed by deep pyrosequencing of PCR products originating from the V1-V2 region of the 16S rRNA gene with bar-coded fusion primers containing Roche-454 A or B Titanium sequencing, followed by a unique 8-base barcode sequence (B) and, finally, the 5′ ends of primer A-8FM (5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGBBBBBBBBAGAGTTTGATCMTGGCTCAG) and of primer B-357R (5′-CCTATCCCCTGTGTGCCTT-GGCAGTCTCAGBBBBBBBBCTGCTGCCTYCCGTA-3′). All PCR reactions were quality controlled for amplicon saturation by quantifying and comparing band intensities of the PCR products after gel electrophoresis with standards using GeneTools software (Syngene). Amplicons from 48 individual samples were pooled in equal amounts, gel-purified, quantified by Pico Green analysis, and used for emulsion PCR (emPCR). After recovery and enrichment for DNA-containing beads, the emPCR products from the 48-sample pools were sequenced on individual regions of 2-region Picotitre plates on a Roche-GS-FLX machine using Titanium sequencing chemistry.