Protease inhibitors and phosphatase inhibitors

Snap-frozen tissues were homogenized using MAGNAlyser beads (Roche Life Science, Penzberg, Germany) in RIPA lysis buffer (Sigma-Aldrich) with protease inhibitors and phosphatase inhibitors (Nacalai Tesque Inc, Kyoto, Japan). The samples were centrifuged for 20 min and the supernatant homogenate collected. Protein concentrations were determined using the Pierce BCA reagent (Thermo Fisher).
Luminex ELISA was carried out for the detection of murine interleukins, chemokines, and growth factors (R&D Systems, MN; LXSAMSM-21). Sample preparation followed the manufacturer’s protocol, and analytes were read using the MAGPIX Platform (Luminex Corporation, TX).
For blotting, 30  μg samples were separated by SDS-PAGE (Bio-Rad Laboratories Inc, CA) and transferred to PVDF membranes (Bio-Rad Laboratories Inc). The PVDF membranes were blocked with 5% blotting grade blocker (Bio-Rad Laboratories Inc) at room temperature (RT) for 1  hr and then incubated with the primary antibodies (anti-myoglobin antibody, 25919S, Cell Signaling Technology) overnight at 4°C. Then, the membranes were washed with TBST at RT and incubated with the secondary antibody for 1  hr. Protein bands were detected with Pierce ECL substrate (Thermo Fisher) according to the manufacturer’s instructions. Data were analyzed with the ChemiDoc Imaging System (Bio-Rad Laboratories Inc).

Cells of high, middle and low densities were seeded in 60-mm dishes overnight, then washed with ice-cold PBS and lysed in lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100) with protease inhibitors and phosphatase inhibitors (Nacalai Tesque, Kyoto, Japan) for 30 min. After centrifugation, the supernatants were collected and protein concentrations were determined using a BCA protein assay kit (Pierce). The protein lysates were subjected to SDS-PAGE. After electrophoresis, the proteins were transferred to a PVDF membrane (Millipore). The membrane was detected with primary and secondary antibodies, and the proteins were visualized by Immobilon Western Chemiluminescent HRP Substrate (Millipore) according to the manufacturer’s instructions. Uncropped scans for the main Western blots were shown in Supplementary information.