Trpv4

Cells were lysed in RIPA buffer containing protease and phosphatase inhibitor cocktails (MilliporeSigma and Roche, Basel, Switzerland). Lysates were loaded into 7.5% precast polyacrylamide gels (Bio-Rad) for electrophoresis. Gels were transferred onto a PVDF membrane and blocked in 5% milk powder in tris-buffered saline (TBS) with 0.1% Tween-20. Membranes were incubated overnight at 4°C with primary antibodies: TRPV4 (1:300; Alomone Labs, Jerusalem, Israel, or 1:300; Biorbyt, San Francisco, CA, United States), and GAPDH (1:5000; Cell Signaling Technology). After incubation, membranes were washed 3× with TBS-Tween-20 for 10 min each, followed by 1 h incubation at room temperature in appropriate secondary antibody, goat anti rabbit (1:5000) conjugated with horseradish peroxidase (Cell Signaling Technology). Signals were detected with Clarity western luminol/enhancer solution and peroxide solution (Bio-Rad laboratories, Hercules CA, United States), and developed with a FluorChem M Simple Imager (Protein Simple, San Jose, CA, United States). Quantification was performed using ImageJ software.

Samples were homogenized in hypotonic homogenization buffer (20 mM Tris-HCl, pH 7.8, 3 mM MgCl₂, 10 mM NaCl, 0.0005 mg/ml, 2 mM sodium vanadate, 20 mM sodium fluoride, 0.5 mM DTT, and 1 mM PMSF) on ice and centrifuged at 15,000 g for 30 min at 4°C to separate cytosolic proteins from intracellular and plasma membranes. The pellet was resuspended in 0.5 M Na₂CO₃, transferred to a 5%/35%/45% sucrose (in Na₂CO₃) flotation gradient and spun at 36,000 rpm for 18 h using a preparative ultracentrifuge model XL-90 (NVT90 rotor; Beckman Coulter Life Sciences). Fractions obtained from the sucrose gradient were diluted in hypotonic buffer and spun at 15,000 g for 30 min at 4°C. Pellets (25 μl) were resuspended in RIPA buffer and 2× Laemmli buffer. About 30 μl of each sample was loaded in 10% SDS-PAGE and transferred to PVDF membranes for 1 h at 220 mA. Nonspecific binding was blocked with 5% nonfat milk and 2% BSA. The samples were incubated overnight at 4°C with TRPV4 (1:500; Alomone Labs), flotillin (1:200; Santa Cruz Biotechnology), Cav-1 (1:1,000; Cell Signaling), and α-SMA (1:500; Sigma-Aldrich) antibodies, followed by anti-mouse (1:5,000; BioRad) or anti-rabbit (1:5,000; Cell Signaling) HRP-conjugated secondary antibodies. The blotted proteins were developed with an enhanced chemiluminescence kit (Thermo Fisher Scientific).

Western blotting was conducted according to standard protocols as previously described42 (link). Protein-extracts (20 μg) of snap-frozen whole LV tissues were run on a 10% or 12% SDS-PAGE gel following by blotting to a nitrocellulose membrane. Western blot band quantifications were performed with Image Lab software (Bio-Rad, Richmond, CA, USA), and the relative values were expressed relative to GAPDH signals. Antibodies against the following proteins were used: TRPV4 (#ACC-034, Alomone labs), Bax (#2772,Cell Signaling), Bcl-2 (#2870, Cell Signaling), Caspase-3 (#19677, Proteintech), GAPDH (Guge), Akt (#4691,Cell Signaling), P-Akt (#4060, Cell Signaling), ERK (#4695, Cell Signaling), P-ERK (#4370, Cell Signaling), GSK-3β (#12456, Cell Signaling), P- GSK-3β (#9331, Cell Signaling). STAT3 (#9132, Cell Signaling) and P-STAT3 (#9131, Cell Signaling).

Lung tissue was isolated, frozen in liquid nitrogen and stored at −80 °C until analysis. For protein extraction, tissue samples were minced and sonicated, and similar to cell monolayers, samples were lysed in RIPA buffer containing protease inhibitors. Lysates were centrifuged and total protein contents were determined by the bicinchoninic acid method. Equal amount of protein lysate samples (20 μg) were fractionated by NU-PAGE Bis-Tris (4–12 %) gel electrophoresis (Novex, San Diego, CA) and transferred to polyvinylidene difluoride membranes. Blots were hybridized with antibody to TRPV4 [1:100] (cat # ACC-034, Alomone, Jerusalem, Israel), phospho (p)-JNK, p-PLA₂, p-p38 or p-ERK [1:100] (all from Cell Signaling Technology, Danvers, MA). Goat anti-rabbit secondary antibodies [1:10,000] were conjugated with horseradish peroxidase; blots were developed with an enhanced chemiluminescence (ECL) detection assay (Amersham Pharmacia Biotech, Piscataway, NJ). Membranes were then stripped and reprobed with antibodies to vinculin, GAPDH, total (t)-JNK, t-PLA₂, t-p38 or t-ERK [1:100] (to control for protein loading) and processed as described before. The intensity of the bands was analyzed by densitometry.

Lysates were separated by 10% SDS-PAGE. The proteins were detected using primary antibodies against TRPV4 (1:500, Alomone Labs, Jerusalem, Israel) and GAPDH (1:1000, Protein tech, Beijing, China). An HRP-labeled IgG was used as the secondary antibody at a 1:1000 dilution, and the bands were visualized using an ECL kit (Beyotime, Shanghai, China) and quantified with Quantity One software (Bio-Rad, Hercules, CA, USA).