Ecltm western blotting analysis system

Fp expression was used as a marker of mitochondrial mass. Cells were homogenized in mannitol buffer with 10% sodium dodecyl sulfate (SDS), 10 mM ethylenediaminetetraacetic acid, 10 mM Ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 63 mM Tris pH 6.8, 6% glycerol, and 50 mM 1,4-dithiothreitol. Samples were heated at 94°C, then run in a 12% acrylamide denaturing gel and transferred to a PVDF membrane. The membrane was incubated with 5% milk in Tris-saline buffer with 0.1% Tween-20 for one hour, followed by incubation with the primary antibody at 4 °C overnight. The incubation with the secondary antibody was performed for one hour at room temperature. The antibodies used were anti-C-II, flavoprotein subunit—Fp (1 μg/μL; Invitrogen, 1:1000 dilution); anti-β actin, (1 μg/mL; ABCAM, 1:500 dilution); anti-rat IgG horseradish peroxidase (HRP) conjugated (0.8 mg/mL; BioRad; 1:50,000 dilution); anti-goat IgG HRP conjugated (400 μg/mL; Santa Cruz, 1:10,000 dilution). The detection was conducted with the ECL^TM Western Blotting Analysis System (GE Healthcare, Piscataway, NJ, USA), followed by autoradiography.

Tumor homogenates, obtained from nude mice were processed as previous described [27 (link)]. Cells were maintained in medium without serum for 24h, exposed to ligands as indicated and then lysed in RIPA buffer containing a mixture of protease inhibitors. Equal amounts of protein extract were resolved on SDS-polyacrylamide gel, transferred to a nitrocellulose membrane (Amersham Biosciences, Italy), probed overnight at 4°C with antibodies against phosphorylated ERK1/2 (E-4), ERK2 (C-14), phosphorylated Akt 1/2/3 (ser 473), Akt (H-136), Elk1 (E-5), Runx1 (A-2), GPER (N-15), β-Actin (AC-15), Ki67 (H-300) ERα (F-10) (Santa Cruz Biotechnology, DBA, Italy) and ERβ (Serotec), and then revealed using the ECL^TM Western Blotting Analysis System (GE Healthcare, Italy).

To construct a cytokine array using a proteome profiler array, we obtained peri-infarcted myocardium tissue after 2,3,5-TTC staining to confirm the peri-infarcted area in the left ventricle 24 h after LAD I/R [32 (link)]. We determined the infarct area in the anterior and posterior sides of each 2 mm-thick slice in the heart tissue between 0 and 8 mm from the apex of the heart. We selected peri-infarcted tissue within 3 mm from the end margin of the infarcted tissue in each 2 mm-thick slice between 0 and 8 mm from the apex. We homogenized the peri-infarcted tissue of the rat heart with a homogenizer (Bertin technologies, Montigny, France) in PBS with a protease inhibitor and performed protein quantitation with a BCA kit following the manufacturer’s protocol. We also conducted Proteome Profiler array using the Mouse Cytokine Array Panel A (R&D Systems, Inc., Minneapolis, MN, USA) as per the manufacturer’s instructions, and visualized the blots using the ECL^TM Western Blotting Analysis System (GE Healthcare, Chicago, IL, USA) and LAS 4000 mini (Fujifilm, Tokyo, Japan). To analyze the array results, we quantized the blots using HLImage++ (Western Vision software, Salt Lake City, UT, USA) (Figure S1) [10 (link)].