Proteinase and phosphatase inhibitors cocktail

After sacrificing animals, tissue samples were collected in RIPA (Sigma‐Aldrich, St. Louis, MO, USA) buffer on ice, with Proteinase and Phosphatase Inhibitors Cocktail (Thermo Fischer Scientific), lysed and homogenised using a Precellys 24 Tissue Homogenizer (Bertin Instruments, 3 × 30 s, 5000 r.p.m.) and protein concentration measured using the Bradford assay (Bio‐Rad). Laemmli buffer (Sigma) treated samples with standardised concentration of 1 µg·µL⁻¹ were incubated at 95 °C for 5 min. Per lane 25 µg of protein was loaded on 10% PAA gel, followed by protein transfer to a methanol activated PVDF membrane (GE Healthcare Life Sciences) over night at 4 °C with constant current of 0.12 A. The membrane was blocked with 5% BSA in TBS‐T (0.01% Tween 20) for 1 h at room temperature. Anti‐MEOX2 1 : 1000 (LS Bio; LS‐B5472) and GAPDH 1 : 1000 (HRP conjugate, Cell signalling; 3683S, Danvers, MA, USA) or HSP90 1 : 1000 (Cell signalling, C45G5) antibodies diluted in Blocking buffer were used for detection of MEOX2 or loading controls respectively, followed by anti‐rabbit or anti‐mouse HRP‐conjugated secondary antibodies 1 : 30 000 (Cytiva, NA934V; Sigma‐Aldrich, GENA931), if required, also diluted in Blocking buffer. The signal was visualised with a chemiluminescent reagent (ECL, GE Healthcare Life Sciences) and imaged (ChemiDoc Imaging System, Bio‐Rad).

Whole-cell lysates were extracted from cultured LX-2 cells using freshly prepared RIPA lysis buffer (Sigma-Aldrich), containing a proteinase and phosphatase inhibitors cocktail (Thermo Fisher Scientific). A Bradford colorimetric assay (Bio-Rad, Hercules, CA, USA) was used to measure the concentration of the protein. Equal amounts of protein (50 µg) were separated on SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% skimmed milk, the blots were incubated overnight at 4 °C with the following primary antibodies: rabbit-polyclonal anti-natriuretic peptide receptor (NPR)1 antibody (1:1000, PA5-29049, Thermo Fisher Scientific); rabbit-polyclonal anti-NPR2 antibody (1:1000, PA5-99836, Thermo Fisher Scientific); and rabbit-monoclonal anti-GAPDH (1:1000, 2118, Cell Signaling Technology, Danvers, MA, USA). The membranes were then washed thrice and incubated with goat anti-rabbit IgG H&L (HRP) (1:2000, ab6721, Abcam) for 1 h at room temperature. The blots were developed with a Clarity Western ECL Substrate (Bio-Rad) using an iBright™ CL1500 Imaging System (Thermo Fisher Scientific).

Cells were lysed with RIPA buffer (Beyotime) supplemented with 1% proteinase and phosphatase inhibitors cocktail (ThermoFisher Scientific). The collected cell lysates were centrifuged for 15 min at 12000rpm (4°C). The supernatant was reserved and the protein concentration was determined with a BCA Protein Assay Kit (ThermoFisher Scientific). 5 × SDS-PAGE loading buffer (Applied Cells, Inc.) was diluted to 1 × with protein sample and heated at 100°C for 8 min. The protein extracts were subjected to appropriate concentrations of SDS–PAGE for electrophoresis and transferred to PVDF membranes (Bio-Rad). Membranes were blocked with 5% bovine serum albumin (ThermoFisher Scientific) for 1 h under room temperature, and then incubated with the primary antibodies overnight at four degrees. Membranes were incubated with secondary HRP-conjugated antibodies (KANGCHEN) at room temperature for 1 h. Before and after the incubation, the membranes were washed five times with TBST and then examined with a Minichemi imaging system (PerkinElmer).