Bradford reagent

Cells were washed with cold PBS, scraped, and lysed in RIPA (Laemmli Sample Buffer). Protein concentrations were measured using Bradford reagent (Sigma-Aldrich). Then, 5% milk was used for blotting. HRP-linked secondary antibodies were used and chemiluminescence was measured after applying ECL Western blotting reagent (AmershamTM, GE Healthcare, Buckinghamshire, HP6 5AY, UK). The same vimentin and Cav-1 antibodies in immunofluorescence were used in Western blot. In addition, vimentin (p-ser39) rabbit monoclonal (dilution 1:1000; Cell signaling, #13614), vimentin (P-ser56) rabbit polyclonal (dilution 1:1000; Cell signaling, Beverly, MA, 01915, USA, #3877), vimentin (P-ser83) rabbit polyclonal (dilution 1:1000; Cell signaling, Beverly, MA, 01915, USA, #3878), and GAPDH mouse polyclonal (dilution 1:1000; Sigma-Aldrich Corp., St. Louis, MO, USA, #G8795) were used.

Astrocyte cultures (or tissue biopsy samples of the injury site) were harvested in lysis buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mm NaCl, 10 mm buffered phosphate, pH 7.2, 2 mm EDTA, 50 mm NaF, 0.2 mm orthovanadate, and protease inhibitor cocktail). Cells were collected with a cell scraper, passed six times through a pipette tip, vortexed, and incubated on ice for 15 min. The lysates were then centrifuged at 20,000 × g for 15 min, and pellets were discarded. Protein concentration of each sample was determined using Bradford Reagent (Sigma). Samples containing 20 mg of protein were boiled in 1× SDS sample buffer, separated by SDS (10%)-PAGE and blotted onto PVDF membranes (Millipore). The membranes were incubated in 5% fat-free milk in TBST (10 mm Tris-HCL, pH 7.4, 150 mm NaCl, and 0.1% Tween 20) for 1 h and then 5% BSA in TBST containing various dilutions of primary antibodies for 18 h at 4°C. The membranes were washed three times with TBST for 5 min each before and after incubation with secondary antibody. The proteins were detected with an appropriate secondary antibody (for 1 h at room temperature) coupled to horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibody and visualized by chemiluminescence according to the manufacturer instructions (Pierce).

Cells were harvested and whole cell lysates were prepared using a RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) with the addition of a protease and phosphatase inhibitor cocktail. Protein content of the lysates was determined using the Bradford reagent (Sigma-Aldrich, St. Louis, MO, USA).

Adipose tissues were homogenized in RIPA buffer containing protease (PMSF, P7626, and Protease Inhibitor Cocktail P8340; Sigma-Aldrich, St. Louis, MO, USA) and phosphatase inhibitors (Pierce Phosphatase Inhibitor Mini Tablets 88667; Thermo Fisher Scientific) and incubated on ice for 1 hour. After centrifugation (10,000 g, 15 minutes, 4°C), the supernatants were collected, and protein concentration was determined with Bradford reagent (B6916; Sigma-Aldrich). Western blot analysis was performed as described by Xue et al. (7 (link)), with goat anti-uncoupling protein (UCP)-1 (sc-6529; Santa Cruz Biotechnology, Dallas, TX, USA) and mouse anti-β-actin (ab6276; Abcam, Cambridge, United Kingdom). The bands were visualized with an Odyssey imaging system (LI-COR Bioscience, Lincoln, NE, USA) with fluorescence-labeled secondary antibodies (IRDye800 and IRDye700; LI-COR), according to the manufacturer’s protocol. UCP1 signals were normalized to β-actin.

Alkaline phosphatase (ALP) activity was determined by colorimetric assay using an ALP kit (Labtest Diagnóstica, Lagoa Santa, MG, Brazil), following manufacturer instructions. BMSCs were osteoinduced under the different conditions stated above for 7 and 14 days and total protein extracts were obtained by scrapping the monolayers in 300 μL of 125 mM Tris-HCl (pH 6.8) 0.5% Triton X-100 buffer. Equal volumes of substrate (22 mmol/L thymolphthalein monophosphate) and protein extracts were mixed and incubated in a water bath at 37°C for 10 min. Reaction was stopped by adding a 250 mmol/L NaOH 94 mmol/L Na₃CO₂ colorimetric solution. The optical density (OD) of the product was measured at 590 nm. Protein concentration of cell extracts was measured with Bradford Reagent (Sigma-Aldrich), and ALP activity was shown as (OD of test sample/OD of standard control sample) × 45/mg of total protein.

The content of free amino acids in freeze-dried tomato fruit was determined by capillary electrophoresis. The experiments were conducted in a fused-silica capillary (Polymicro Technologies, Phoenix, AZ, USA) using a G7100A Capillary Electrophoresis Instrument (Agilent Technologies, Waldbronn, Germany) with a contactless conductivity detector [39 (link)]. Protein content was analyzed with Bradford reagent (Sigma-Aldrich, St. Louis, USA) [40 (link)].