Hyperfilm

Ascending aortic medial tissue from 6-mo-old wild-type (male n = 6; female n = 6) and Marfan (male n = 6; female n = 6) mice was homogenized using a bullet blender, ø 0.9–2 and 3 mm stainless steel beads (Next Advance, NY, USA) in 300 μl radioimmunoprecipitation assay buffer (RIPA, 10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 7.5, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium desoxycholate, 0.1% SDS, 140 mM NaCl) and protease inhibitors. Protein concentrations were assessed using a colorimetric assay for protein concentration (Biorad, CA, USA). Twenty micrograms of protein per sample were analyzed by 10% (v/v) SDS-PAGE and transferred to nitrocellulose membranes. Primary antibodies to COX-1 (Santa Cruz, CA, USA), COX-2, eNOS, (p)eNOS (BD Transduction Laboratories, CA, USA), iNOS (Thermo Fisher, MA, USA) and actin (Sigma Aldrich, MO, USA) were incubated overnight in TBS 1% BSA. Protein bands were revealed using secondary IgG HRP conjugates (Promega, WI, USA) and Luminol Reagent together with Hyperfilm (Amersham Pharmacia Biotech, Uppsala, Sweden). Band intensities were measured by densitometry scanning using ImageJ software (National Institute of Health, Bethesda, MD, USA). Band intensities were relativized against actin as a loading control and the values of all groups were normalized for WT male average values.

Equal amounts of protein (10 μg) were resolved on 10% Criterion XT Bis-Tris Precast Gels (Bio-Rad, Hercules, CA) and transferred to PVDF membranes. Membranes were blocked with TBS-T (TBS and 0.5% Tween 20) containing 5% (w/v) non-fat dry milk or 5% BSA (depending on the primary antibody) for 30 minutes to 1 h and washed with TBS-T. Membranes were then incubated overnight at 4°C with different antibodies including FASN (clone 23, BD Biosciences, Pharmingen), p21 (F-5, Santa Cruz Biotechnologies, Santa Cruz, CA) and AMPK, p-AMPK, ACC and p-ACC (Cell Signaling Technology, Danvers, MA). After a further 3 washes with TBS-T, membranes were incubated with horseradish peroxidase-linked secondary antibodies. Blots were then stripped and re-probed with a monoclonal antibody to β-actin and horseradish peroxidase-linked goat anti-mouse IgG secondary antibody. Proteins were detected by an enhanced chemoluminescence reaction using Hyperfilm (Amersham-Pharmacia, Piscataway, NJ).

Neural progenitor cells or tissues from different brain regions were homogenized in 0.7% NP40, 50 mM Tris-HCl (pH 8.0), 0.1 mM EDTA (pH 8.0), 250 mM NaCl, 10% glycerol, 0.2 mM Na₃VO₄, 1 mM PMSF, 10 mM DTT, 2 μg/ml Aprotinin and 1 μg/ml Pepstatin and centrifuged at 17,900 × g for 15 min. at 4°C. The protein concentration in the supernatants was determined using the BCA kit (Sigma-Aldrich). Protein extracts were resolved on 12% SDS-PAGE and transferred onto nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany) as described previously [10 (link)]. Antibodies for Wetern blots were as follows: rabbit anti-phospho smad 2, 1:500 (Cell Signaling); mouse anti-smad 2 (1:500; Cell Signaling); mouse anti-TGFbRII (1:500; Santa Cruz Biotechnology, USA); rabbit anti-TGFbR1 (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA); rabbit anti-actin (1:5000; Sigma-Aldrich). Secondary goat antimouse or anti-rabbit IgG-HRP antibodies (1:10,000; Dianova) and detection was performed with the ECL plus chemiluminescence system (Amersham/Pharmacia, Freiburg, Germany) and exposed to Hyperfilm (Amersham Pharmacia). Blots were stripped and reprobed as described [23 (link)].

Mouse skin tissues (at the junction of normal and scar) of each group were isolated for western blot analysis. Samples were homogenized and proteins were extracted using RIPA buffer according to manufacturer’s protocol (Cell Signaling Technology, USA, for 30 minutes). Proteins were separated by SDS-PAGE (10% gel) and transferred onto polyvinylidene difluoride (PVDF) membrane (Sigma-Aldrich, USA). The membrane was blocked in 5% bovine serum albumin (BSA) for 2 hours at room temperature. Then, the blots were incubated with p-p38 (Cell Signaling Technology, USA, 1:1000, 4 °C overnight) or KGF-2 antibodies (R&D Systems, USA, 1 µg/mL, 4 °C overnight) in 5% BSA solution, followed by incubation with secondary antibodies conjugated with horseradish peroxidase for 1 hour. Signals were visualized with enhanced chemiluminescence (ECL) and exposure to Hyperfilm (Amersham Biosciences, UK). β-actin expression was served as a loading control.