Complete protease inhibitor cocktail

Whole-lung extracts were prepared from male mice lungs with nine volumes of ice-cold buffer (pH 7.4:5 mM phosphate buffer containing a complete protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan)) using Biomasher SP (Nippi Inc., Tokyo, Japan) for the thiobarbituric acid-reactive substances (TBA-RS) assay. Cytoplasmic and nuclear extracts were prepared using NE-PER™ nuclear and cytoplasmic extraction reagents (the protease and phosphatase inhibitors were added to CER I and NER) (Thermo Scientific, Rockford, IL, USA) using Biomasher II, according to the manufacturer’s instructions. Membrane extracts were prepared using the Mem-PER™ Plus Membrane Protein Extraction Kit (Thermo Scientific, Rockford, IL, USA) and Biomasher II according to the manufacturer’s instructions.

Excised gastrocnemius muscles were immediately frozen in liquid nitrogen, and total muscle protein was extracted with lysis buffer containing 50 mM HEPES (pH: 7.6), 150 mM NaCl, 10 mM EDTA, 10 mM Na₄P₂O₇, 10 mM NaF, 2 mM Na₃VO₄, 1% (vol/vol) NP‐40, 1% (vol/vol) Na‐deoxycholate, 0.2% (wt/vol) SDS, and 1% (vol/vol) complete protease inhibitor cocktail (Nacalai Tesque Inc.). Protein concentrations were measured using a Protein Assay Bicinchoninate Kit (Nacalai Tesque Inc.). Before SDS‐PAGE, an aliquot of the extracted protein solution was mixed with equal volume of sample loading buffer containing 1% (vol/vol) 2‐mercaptoethanol, 4% (wt/vol) SDS, 125 mM of Tris‐HCl (pH: 6.8), 10% (wt/vol) sucrose, and 0.01% (wt/vol) bromophenol blue. The mixture was then heated at 97°C for 3 min. Ten micrograms of protein was separated on an SDS‐polyacrylamide gel and electrically transferred to an ImmunoBlot PVDF membrane (Bio‐Rad Laboratories). The blot was blocked by Blocking One (Nakalai Tesque Inc.) for 1 h at room temperature and incubated with primary antibodies overnight at 4℃ in TBS containing 0.1% Tween 20. Signals were detected using the Immunostar Zeta or LD (Wako Chemicals), quantified by C‐Digit (LI‐COR Biosciences), and expressed as arbitrary units. The expression levels of each protein were normalized to those of glyceraldehyde 3‐phosphate dehydrogenase.

LECs were pre-cultured with FBS-free and supplement-free EGM2 medium (Lonza) for 24 h. Cells were then treated with CCN2-containing FBS-free and supplement-free EGM2 medium and cultured for the appropriate time. Cells were washed with PBS and lysed in RIPA Buffer (Merck) containing cOmplete protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Nacalai, Kyoto, Japan). Protein concentration was determined with the BCA protein assay kit (Thermo). Protein samples (5–20 µg) were separated with SDS-PAGE and transferred to PVDF membranes (Invitrogen). The membranes were blocked in Blocking-one (Nacalai) for 30 min and cut prior to hybridization with antibodies, then incubated with primary antibodies at 4 °C overnight. The primary antibodies are listed in Supplementary Table S3. After three washes with PBS containing 0.1% Tween-20, the membrane was incubated with HRP-conjugated anti-rabbit IgG or HRP-conjugated anti-rat IgG secondary antibody (Cell Signaling Technology, Danvers, MA, USA) for 2 h. After three washes with PBS containing 0.1% Tween-20, signals were visualized using ECL Prime (GE Healthcare, Chicago, IL, USA) and an image analyzer 680 (GE Healthcare). Signals were quantified using ImageQuant TL 8.1 software (GE Healthcare).