Imagequant tl

Total RNA (15 µg) was loaded on a 10% TBE-UREA gel. After electrophoresis, gel was stained with SYBR gold for visualization of equal loading. Gel was transferred onto a positively charged Nylon membrane for 1 hr at 250 mA. After UV-crosslinking, the membrane was pre-hybridized for 4 hr at 40°C in 1xSSC, 1%SDS (w/v) and 100 mg/ml single-stranded DNA (Sigma). Radioactively labelled probes corresponding to the highly expressed ESCs miRNAs miR-130–3 p, miR-293–3 p, and miR-294–3 p were synthesized using the mirVana miRNA Probe Construction Kit (Ambion) and hybridized overnight in 1xSSC, 1%SDS (w/v) and 100 mg/ml ssDNA. After hybridization, membranes were washed four times at 40°C in 0.2xSSC and 0.2%SDS (w/v) for 30 min each. Blots were analysed using a PhosphorImager (Molecular Dynamics) and ImageQuant TL software for quantification. Oligonucleotides used are listed in Supplementary file 1.

Image analysis (ImageQuant TL, Molecular Dynamics) was performed followed by statistical analysis by applying unpaired Student’s t-test (between Col6a1^−/− and wt) or independent one-way ANOVA and Tukey (among the 3 timepoints), n = 2, p < 0.05. Band intensities were normalized against the total amount of proteins stained by Sypro Ruby.

Protein extracts (50 μg) from samples analyzed by 2D-DIGE were loaded in triplicate and resolved on 5-12% polyacrylamide gels, according to protein molecular weight. Blots were incubated with rabbit or goat polyclonal primary antibodies (Santa Cruz Biotechnology except when differently stated) as follows: anti-NADH dehydrogenase (ubiquinone) Fe-S protein 1 (NDUFS1), 1:500; anti-peroxisome proliferator-activated receptor alpha (PPARα), 1:500; anti-ATP synthase 5A (ATP5A1), 1:500; 1:500; anti-isocitrate dehydrogenase 2 (IDH2), 1:500; anti-glutamic-oxaloacetic transaminase 2, (GOT2, Bioss Antibodies), 1:1000; anti-fumarate hydratase (FH), 1:500. After washing, membranes were incubated with anti-rabbit (GE Healthcare) or anti-goat (Santa Cruz Biotechnology) secondary antibodies conjugated with horseradish peroxidase. Signals were visualized by chemiluminescence using the ECL Plus detection kit. Image analysis (Image Quant TL; Molecular Dynamics, Sunnyvale, CA, USA) was performed and followed by statistical analysis (paired Student’s t test, P < 0.05). Data were normalized against the total amount of proteins stained by Sypro Ruby (Molecular Probes) (Supplementary Fig 1).

Aliquots of TS and TI fractions were used for phospholipids phosphorus determination by the Bartlett procedure. Then, 7 μg of phosphorus from each sample were subjected to lipid extraction according to Tettamanti’s protocol (Tettamanti et al., 1973 (link)). The lipid extracts in the organic phases were analyzed by HPTLC. In particular, for phospholipid and cholesterol analysis the solvent system was chloroform/methanol/acetic acid/water (60/45/4/2, vol/vol/vol/vol). Phospholipids and cholesterol were visualized on the same HPTLC plate with anisaldehyde reagent (0.5 ml anisaldehyde, 1 ml 97% sulfuric acid in 50 ml glacial acetic acid). After heating the plate at 180°C for 5 min, the plates were scanned and analyzed with ImageQuant^TM TL (GE Healthcare Life Sciences), program 1D gel analysis.

The protein content of TS and TI fractions was quantified by micro-BCA assay (Merck KGaA, Darmstadt, Germany). Thereafter, 15 μg of proteins were loaded onto a 12% polyacrylamide gel prior to SDS-PAGE analysis. Subsequently, proteins were transferred to membranes that were stained with Ponceau S to assess proper transfer. Blots were washed with TBS and blocked for 1 h in TBS-T/skimmed milk. After blocking, blots were incubated overnight with the primary antibody diluted in TBS-T/skimmed milk [anti-CAV-1 1:1,000, anti-CD55 1:200], and then for 1.5 h with HRP-conjugated anti-rabbit IgG (5,000-fold diluted in TBS-T/milk). TS and TI fractions were obtained from three independent experiments. Proteins were detected by ECL with the Super Signal detection kit (Thermo Scientific) and analyzed with ImageQuant^TM TL (GE Healthcare Life Sciences), program 1D gel analysis.

Equal amount proteins from thoracic aorta and mesenteric arteries were separated by SDS/PAGE and transferred to nitrocellulose membrane, blocked with 5% non-fat dry milk or bovine serum albumin (BSA) in tris-buffered saline tween-20. The membranes were incubated overnight at 4 °C with antibodies as previously described^{19 (link)}. Gels were analysed and quantified by ImageQuantTM TL instrument (GE Healthcare, Buckinghamshire, England).

BM supernatants were generated by crushing 2 tibias and 1 femur in the presence of 400 μl of PBS in a sterile mortar. Samples were centrifuged (600 × g for 10 min), protein levels were quantified using Pierce™ BCA Protein Assay Kit and 100 μg of total protein were used. Cytokine levels were determined using Proteome Profiler Mouse XL Cytokine Array (R&D systems) according to the manufacturer’s instruction. Quantification was done by ImageQuant(TM) TL (GE Healthcare, Chicago, IL, USA) and the cytokine levels were expressed as arbitrary units.