Ecl detection system

After the desired treatments, whole cell lysates were prepared using ProEXTM CETi Lysis Buffer (with protease and phosphatase inhibitors, TransLab, Daejeon, Korea) and the concentrations of protein were determined using a BioRad protein assay kit (BioRad Laboratories, Hercules, CA, USA). The protein samples were subjected to SDS-PAGE, transferred to the 8% nitrocellulose membrane, and then blocked with 5% bovine serum albumin in TBST (10 mM Tris, 100 mM NaCl, and 0.1% Tween 20) for 40 min at room temperature. Next, the membranes were probed with specific primary antibodies at 4 °C overnight and subsequently probed with peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membranes were visualized using an ECL detection system (Roche Applied Science, Indianapolis, IN, USA) with iBright chemi-doc fl000 from Thermo Fisher Scientific (Thermo Fisher Scientific, Cleveland, OH, USA). For each analysis, at least three biological replicates were performed.

Pellets (n = 1 per donor and group) were digested with pepsin solution [2.5 mg pepsin/ml pepsin buffer (0.5 M acetic acid, 0.2 M NaCl)] for at least 16 hours. The pH was then adjusted to neutral pH 7 with 1 M Tris Base prior to extraction of the collagens with 4.5 M NaCl (overnight at 4 °C, both Roth, Karlsruhe, Germany). After centrifugation, the pellets were resuspended in 400 μl precipitation-buffer (0.1 M Tris Base, 0.4 M NaCl) and the collagens precipitated for 4 hours at −20 °C with 100% ethanol. After centrifugation, the pellets were resuspended in lysis buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100). The collagen type II content was measured by native type II collagen detection ELISA kit (Chondrex, Redmond, USA) according to manufacturer’s instructions.
For collagen type II and X Western blotting, pellet lysates were separated by denaturing SDS-PAGE and proteins blotted onto a nitrocellulose membrane. The lower part of the membrane was incubated with mouse anti-human collagen type X antibody (Quartett) and the upper part with mouse anti-human collagen type II antibody (ICN Biomedicals). Bands were visualized with peroxidase-coupled goat anti-mouse antibody using the ECL detection system (Roche).

For western blot determinations of GGT, isolated monocytes, lymphocytes, platelets and cultured endothelial HMEC-1 cells—harvested in hypotonic lysis buffer (10 mM Tris–HCl, pH 7.8)—or plaque derived exosomes were used. Non-adherent lymphocytes were isolated from plated PBMCs, whereas platelets were obtained from buffy-coat derived platelet concentrates. All samples were thoroughly washed in order to remove contaminating plasma GGT. Endothelial HMEC-1 cells were grown in MCDB 131 medium (Life Technologies) supplemented with 1 µg/ml hydrocortisone, 10 ng/ml EGF (Life Technologies) and 10 % v/v fetal calf serum, and cultured at 37 °C in a 5 %/95 % CO₂/air atmosphere.
All samples were separated by 8 % SDS-PAGE [37 (link)] and incubated with rabbit anti-GGT IgG directed against the C-terminal 20 amino acids of human GGT heavy chain prepared as described [36 (link)]. Visualization of protein bands was obtained using a horseradish peroxidase-conjugated anti-rabbit IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the ECL detection system (Roche, Basel, Switzerland). Bands were analyzed with a Bio-Rad ChemiDoc apparatus equipped with the QuantityOne software.

The protein expression was validated as previously described [24 (link)]. Briefly, whole cell lysates (WCL) were prepared using radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 25 mM NaF, 1 mM dithiothreitol (DTT), and 20 mM ethyleneglycoltetraacetic acid (EGTA) supplemented with protease inhibitors) and the protein concentrations were determined using a BioRad protein assay kit (BioRad Laboratories, Hercules, CA, USA). Protein samples were subjected to SDS-PAGE, transferred to a nitrocellulose membrane and then blocked with 5% bovine serum albumin in tris-buffered saline with Tween 20 (TBST) (10 mM Tris, 100 mM NaCl, and 0.1% Tween 20). Next, membranes were probed with specific primary antibodies and subsequently peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membranes were analyzed using an ECL detection system (Roche Applied Science, Indianapolis, IN, USA) with iBright chemi-doc fl000 from Thermo Fisher Scientific.

The protein levels of CD63, a protein involved in exosomes biogenesis [21 (link)], were evaluated by western blotting analysis. For this purpose, 50 mg of lung tissue were lysed in RIPA lysis buffer supplemented with a protease inhibitor cocktail (Sigma Aldrich) and then homogenized with an electric homogenizer. The lysates were obtained by centrifugation at 12,000g for 20 min at 4 °C. In brief, 100 μg of each sample lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to 0.2-μm polyvinylidene difluoride (PVDF, 249 Millipore) membranes. After blocking with 5% skim milk (Gibco), membranes were incubated with primary anti-human CD63 antibody (Cat no: sc-5275) overnight at 4 °C followed by incubated with secondary antibody (goat anti-mouse IgG-HRP: DB9571, Iran) 1 h at 37 °C. Immunoblotting bands were visualized using a chemiluminescence (ECL) detection system according to the manufacturer’s instructions (Roche). β-actin (Cat no: sc-47778) was kept as a housekeeping protein for normalization. Relative protein levels were calculated using NIH ImageJ software ver. 1.44p.

Whole-cell extracts were subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS–PAGE), transferred to nitrocellulose membranes, and blotted following standard procedures. Membranes were probed with the following primary antibodies: rabbit polyclonal anti-Tom20 (clone FL-145, no. 11415) from Santa Cruz; mouse monoclonal anti-c-myc (clone 9B11, no. 2276S) and rabbit monoclonal anti-HA (C29F4; no. 3724) from Cell Signaling and rat anti-tubulin YL1/2 (ab6160) from Abcam. Secondary antibodies specific for mouse, rabbit, or rat IgG conjugated with peroxidase for enhanced chemiluminescence (ECL) detection were purchased from Sigma-Aldrich. Immunoblot signals were detected using ECL detection reagents and acquired in an ECL™ Detection System (Roche).