Enhanced chemi luminescence (ecl)

For the myelin basic protein (MBP) analysis a total 5 µg of spinal cord samples (T9-T11), from 3 experimental groups (naïve, saline and DHA), at four time points (4 h-24 h-7 days-30 days) were used (n = 3 mice/group/time point). Membranes were incubated overnight at 4 °C with the following primary antibody: Myelin Basic Protein (MBP101, Abcam Cat# ab62631, RRID:AB_956157) and β-actin (AM1829b, Abgent Cat# AM1829b, RRID: AB_10664137). Antibody binding was revealed by using enhanced chemiluminescence (ECL) (Euroclone, Pero, Mi, Italy). Luminescent bands were imaged with autoradiography (X-ray) films (UltraCruz; Santa Cruz Biotechnolgy) and then scanning into a digital format. The βactin bands intensity was used as a control for equal protein loading and measured for densitometric analysis using ImageJ 1.49r software (Wayne Rasband, National Institutes of Health).

Cells from GIST 430-, 882- and T1-derived tumors were extracted with RIPA buffer (1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris–HCl pH 7, 0.4 mM Na₃VO₄, inhibitor mix). Cell lysates were centrifuged at 13,000×g for 10 min, and the supernatants were collected and assayed for protein concentration with the Bio-Rad protein assay method (Bio-Rad, Hercules, CA, USA). Total cell lysates were separated by SDS-PAGE under reducing conditions, transferred to nitrocellulose, and immunoblotted overnight with primary antibodies against vinculin (loading control), VEGFR2, and VEGFR3 at 4 °C. Mouse monoclonal antibody against vinculin was produced at the Molecular Biotechnology Center (MBC), while antibodies against VEGFR2 and VEGFR3 were purchased from Cell Signaling (Beverly, MA, USA). Blots were incubated with mouse or rabbit horseradish peroxidase conjugated secondary antibodies for 1 h at room temperature. ECL (Euroclone) was used to detect chemoluminescent signals. Protein band intensities were measured by a scanning densitometer (Quantity One; PDI Inc., Huntington, NY, USA).

For pAMPKα analysis, a total of 50 µg of sciatic nerves (pool of 3 nerves from 3 different animals for each single experiment, and 50 ug of total protein lysate for each gel was loaded), from 3 experimental groups (naïve, saline and metformin), at four time points post CCI (6 h-24 h-3 days-7 days) were used (n = 3 mice/group/time point). Membranes were incubated overnight at 4 °C with the following primary antibody: pAMPKα (Phospho-AMPKα-T172 polyclonal antibody; Elabscience) and β-actin (AM1829b, beta Actin, monoclonal antibody; Abgent). Antibody binding was revealed by using enhanced chemiluminescence (ECL) (Euroclone, Pero, Mi, Italy). Luminescent bands were imaged with autoradiography (X-ray) films (UltraCruz; Santa Cruz Biotechnology, Dallas, TX, USA) and then scanned into a digital format. The β-actin band intensities were used as a control for equal protein loading and measured for densitometric analysis (AMPK/Actin) using ImageJ 1.49r software (National Institutes of Health, USA).

Astrocytes and neurons were lysed with a buffer containing 1% SDS, 2 mM EDTA pH 7.4, 10 mM Tris‐HCl pH 7.4 and proteases inhibitor (1:500). A modified version of the Laemmli buffer was then added to a final 1× concentration (15% SDS, 575 mM sucrose, 325 mM Tris‐HCl pH 6.8, 0.5% β‐mercaptoethanol, 0.01% bromo‐phenol blue). EVs released from 20 × 10⁶ astrocytes were lysed with the same Laemmli buffer. 10 μg of total lysates of neurons or astrocytes and the whole pellet of EVs were loaded on a polyacrylamide gel. Proteins were then separated by electrophoresis, blotted on nitrocellulose membrane and probed using mouse anti‐PrP (1:1000; W226; Dr Lothar Stitz, Institute of Immunology, Tuebingen, Germany), rabbit anti‐Alix (1:500; Covalab, Villeurbanne, France), mouse anti‐Flotillin (1:1000, BD Biosciences, CA, USA), rabbit anti‐Annexin A2 (1:5000, Abcam, UK), rabbit anti‐Tom‐20 (1:500; Santa Cruz Biotechnology, CA, USA), mouse anti‐GS28 (1:1000; BD Biosciences, Franklin Lakes, NJ, USA), mouse anti‐Actin (1:500; Sigma, St. Louis, MO, USA), mouse anti‐βIII‐tubulin (1:4000; Promega, Madison, WI, USA) or Rabbit anti‐GAPDH (1:2000; Synaptic Systems, Gottingen, Germany) antibodies. Photographic development was by chemiluminescence (ECL, Euroclone or FEMTO, Thermo Scientific, MA, USA) according to the manufacturer's instructions. Western blot bands were quantified by ImageJ software.

Cells were resuspended in 3–5 volumes of RIPA buffer, containing NP-40 (1%), sodium deoxycholate (0.5%), sodium dodecyl sulfate (SDS) (0.1%), aprotinin (10 μg/ml), leupeptin (1 mM), and PMSF (1 mM) and incubated on ice for 1 h. After centrifugation (18,000 × g for 15 min at 4°C), supernatant was collected and protein concentration was determined by Bradford method. Laemmli buffer 4 × was added to each sample before boiling at 95°C for 5 min. Typically, a quantity of 20–40 μg of total extracts was applied to polyacrylamide gel (Bio Rad Laboratories); thereafter, proteins were divided by weight in SDS–polyacrylamide gel electrophoresis (PAGE) and moved on nitrocellulose membrane (Sigma-Aldrich) using Mini Trans-Blot BioRad (Bio Rad Laboratories). In order to fill uncovered spots, obtained films were incubated in nonfat milk (5% w/v) and then blotted overnight with primary antibody according to the experimental procedures. The next day, HRP-conjugated goat anti-rabbit or anti-mouse was used to detect protein–antibody complexes. Each incubation was preceded and followed by 5 min wash with TBS Tween-20 (Thermo Fisher Scientific) for three times. Finally, nitrocellulose membranes were detected by standard chemical luminescence method ECL (Euroclone). Immunoblotting signals were captured using Chemi-Doc XRS (Bio-Rad Laboratories) and quantitatively analyzed by ImageJ (NIH, Bethesda).

Typically, 15 to 30 μg of total cellular protein was loaded in polyacrylamide gel (Bio-Rad Laboratories) and separated by SDS-PAGE. Thereafter, proteins were transferred onto the nitrocellulose membrane (Sigma-Aldrich) using Mini Trans-Blot (Bio-Rad Laboratories). Before incubating overnight at 4°C using specific primary antibodies, membranes were blocked in no-fat milk (5% w/v) for 1 hour. The next day, horseradish peroxidase- (HRP-) conjugated goat anti-rabbit or anti-mouse antibodies were added to the membranes and kept for 1 hour at room temperature. TBS Tween-20 (Thermo Fisher Scientific) was used to wash the membranes (three times) before and after each incubation procedure. Enhanced chemiluminescence (ECL) (Euroclone) was employed to detect the HRP secondary antibody signal. To conclude, protein bands were detected with ChemiDoc XRS (Bio-Rad).