Ponceau red

Samples (N = 4 per group) from the mid-belly of supraspinatus muscles (50 mg) were homogenized in tissue-TEK lysis buffer (Invitrogen), and protein concentration was measured using BCA protein assay (Thermo Fisher Scientific). In a 4-15% gradient gel, 20 μg of protein were loaded, and separated proteins were transferred to a nitrocellulose membrane. Membranes were stained with Ponceau red (Sigma) to confirm successful transfer of protein and equal loading of lanes. Membranes were then blocked in 5% milk and incubated overnight in primary Anti-Ubiquitin antibody (Sigma, cat number U0508) at a 1:500 dilution. Membranes were visualized after incubation with HRP-conjugated goat antibodies and ECL substrate (Thermo Fisher Scientific). Bands were quantified using ImageJ software and normalized to total protein.

The total protein content of extracts from young and senescent eMVs (performed using CytoBuster Protein Extraction Reagent lysis buffer (Millipore)), which contains a protease and a phosphatase inhibitor cocktail (Roche), was quantified using a BCA Protein Assay Kit (Pierce), employing BSA as the standard. Briefly, equal amounts of protein (25 μg protein/lane) were diluted with a reducing sample buffer and separated by 4–20% Mini-PROTEAN® TGX™ Precast Protein Gels under reducing conditions. These proteins were then transferred onto nitrocellulose membranes (BioRad), blocked with TBS containing 0.1% Tween 20 and 5% dry nonfat milk for 1 h at room temperature, and incubated in the same buffer with different primary antibodies (anti-6PGL, Santa Cruz, sc-398833, dilution 1 : 500, 28 kDa; anti-GK, Santa Cruz, sc-393555, dilution 1/250, 61 kDa; anti-PSPH, Santa Cruz, sc-271421, dilution 1/250, 25 kDa). After washing with TBST, the membranes were incubated with Novex horseradish peroxidase-conjugated secondary antibodies followed by 2 additional washing steps with TBST. Bands were visualized with Luminata Crescendo Western HRP substrate (Millipore). Ponceau red (Sigma) staining was used as a loading control. Bands were quantified using Image J software (NIH) and normalized to Ponceau red.

[7,8-³H]Dopamine (21.2 Ci/mmol (0.784 TBq/mmol)) and [1-7,8-³H]noradrenaline (12.1 Ci/mmol (0.448 TBq/mmol)) were purchased from Perkin Elmer, Monza, Italy. Nicotine hydrogen tartrate salt, choline, 4-aminopyridine, 6-nitroquipazine, desipramine, beta-amyloid (40-1), horseradish peroxidase-conjugated anti-mouse secondary antibody, Ponceau Red were from Sigma (Sigma-Aldrich, St Louis, MO, USA); Monoclonal antibody 6E10 was from Covance ImmunoTechnologies, Dedham, MA; 5-iodo-A-85380, dihydro-β-erythroidine, α-conotoxin MII, oxotremorine sesquifumarate, desformylflustrabromine hydrochloride, galantamine hydrobromide, SR 165845, beta-amyloid (1-40) were from Tocris (Tocris Bioscience, Bristol, UK).

Adherent cells were washed in PBS and lysed directly in preheated Laemmli buffer devoid of bromophenol blue and beta-mercaptoethanol. Lysates were transferred to tubes, boiled for 15 min, and the protein concentration was determined using BCA protein quantification kit (Pierce). After normalization of protein concentrations and addition of bromophenol blue and beta-mercaptoethanol, samples were boiled again for 10 min, and 15 to 50 μg of protein was loaded per sample in standard SDS-PAGE gels. Proteins were transferred to nitrocellulose membranes (Bio-Rad), visualized with Ponceau Red (Sigma), and the membranes were cut horizontally such that different portions of the same membrane could be incubated with the appropriate antibodies. Membranes were blocked with 5% BSA for 30 min and incubated with primary antibody overnight at 4 °C. After extensive washing in TBST buffer, 1 h incubation in appropriate HRP-conjugated secondary antibodies (Sigma), and another round of washing, membranes were incubated with ECL Prime (Amersham), and the signal was detected using a Chemidoc MP Imaging System (Bio-Rad). Signals were quantified using ImageJ software.

Conditioned media were concentrated by TCA precipitation and solubilized in Laemmli sample buffer. An equal amount of proteins were separated under reducing conditions on a 10% or 12% polyacrylamide gel. Resolved proteins were transferred onto a Hybond-C nitrocellulose membrane (GE Healthcare, München, Germany) in a semidry transfer system. The transfer efficiency was assessed by staining the membrane with Ponceau Red (Sigma-Aldrich, Diesenhofen, Germany). Membranes were blocked for 1 h with 5% milk powder and incubated overnight at 4 °C with the primary antibody. Primary antibodies used were anti-ADAM9, anti-decorin, (AF949 and MAB143, respectively, all from R&D Systems, Wiesbaden, Germany), anti-collagen type I (1:500; 20315035505 from Quartett, Berlin, Germany), anti-fibronectin and anti-actin (F3648 and A2668, both 1:1000; from Sigma Aldrich, Diesenhofen, Germany), and anti-MMP-13 (sc-30073, from Santa Cruz, Heidelberg, Germany; 1:1000), anti-MMP-14 (1:500) [18 (link)]. After washing, membranes were incubated with HRP-labeled secondary antibody (from Dako, Hamburg, Germany) for 1 h at room temperature. Bound secondary antibodies were detected by ECL^® system (Thermo Scientific, Bonn, Germany) and the membranes exposed to X-ray films (Thermo Fisher, Karlsruhe, Germany).

For assessment of choroidal protein levels, eyes were enucleated from mice 3 days after burn. RIPA buffer with anti-protease and anti-phosphatase (BioRad) was freshly prepared to manually with a piston to homogenize tissues and for cells lysis. Protein concentration was assessed by BCA assay (Sigma-Aldrich, Oakville, CA), and 30μg of protein analyzed for each condition by standard SDS-PAGE technique using Bis-Acrylamide gel 10% or 12.5% depending of protein size. Total protein transfer on nitrocellulose or PVDF membranes (Bio-Rad, Mississauga, ON, CA) was evaluated with Ponceau Red (Sigma-Aldrich, Oakville, CA). Antibody solutions and dilutions were prepared as per manufacturers’ recommendations.