Tissue protein extraction buffer

To analyse cytokine levels, the right lung tissues were homogenized with 50 mg/mL tissue protein extraction buffer (ThermoFisher Scientific Inc., Rockford, IL, USA) using a tissue homogenizer (Biospec Products, Bartlesville, OK, USA). After incubation for 30 min on ice, homogenates were centrifuged at 14,000 rpm for 10 min. Supernatants of lung homogenates were collected and stored at −80°C for measurement of cytokine levels. Concentrations of IL-17, TGF-β1, IL-1β, TNF-α, and IL-6 in lung homogenate were measured using an enzyme-linked immunosorbent assay (ELISA, R&D Systems, San Diego, CA, USA) according to the manufacturer’s instructions.

The expressions of T-bet and GATA3 in lung tissues were detected by western blot. We homogenized the lung tissues in tissue protein extraction buffer (Thermo Fisher Scientific, Rockford, IL) in the presence of protease inhibitor cocktail to obtain extracts of lung proteins. The proteins were fractionated on 10% SDS-polyacrylamide gels. They were transferred to polyvinylidene fluoride membranes, and the membranes were incubated for 1h in 5% skim milk in TBS-T buffer (0.1 M Tris-HCl, pH 7.4, 0.9% NaCl, 0.05% Tween-20) to block non-specific binding and was then incubated with primary antibodies for overnight at 4°C. Antibody against GATA3, T-bet, GAPDH and secondary antibodies were obtained from Santa Cruz (Dallas, TX). The immunoblots were washed and incubated with appropriate secondary antibodies for 1 h. The blot was developed using an ECL kit (Thermo Fisher Scientific).

Mice cornea were isolated and weighed. Due to the tiny size of mouse cornea, the cornea from four mice treated in the same group were collected and lysed to get enough amount of protein lysate for examination. Cornea from two batches of animal test were examined. The total proteins from these tissues were extracted with tissue protein extraction buffer (Thermo Fisher Scientific, IL, USA). Bead beater-type homogenizer was operated at 2600 rpm, followed by centrifugation at 10,000 g for 3 min at 4 °C, then the supernatants were collected. Total protein was quantified by Bradford assay (p010, GeneCopoeia, Rockville, MD, USA). Angiogenesis cytokines (MMP-2, MMP-9 and VEGF) content in each group were measured by Quantikine^® ELISA kit, Mouse VEGF/total MMP-9/MMP-2 Immunoassay (R&D Systems, Inc. Minneapolis, MN, USA) in triplicate. This experiment was conducted according to manufacturer’s protocol.

The total proteins were extracted using tissue protein extraction buffer (Thermo Scientific, Ref: 78510) containing proteinase inhibitors (Complete Mini, Roche, Ref: 11836170001, Roche, Basel, Switzerland) according to the manufacturer’s instructions. Briefly, HBMECs were washed twice with DPBS solution and then incubated in the lysis buffer for 10 min on ice and vortexed vigorously. The samples were spun down at 10,000 g and the supernatant was stored at −80 °C for downstream analysis. The extracted protein was separated on a NuPAGE 4 to a 12% gradient gel (Invitrogen, Ref: NP0322). Primary antibodies (Phospho-CRKL (Tyr207) (E9A1U) Cell Signaling #34940, 1:800; CRKL (D4G7G) Cell Signaling #38710, 1:800, Cell Signaling Technology, Danvers, MA, USA) were bound by HRP- conjugated secondary antibodies and visualized using the Azure c600 imaging system (Azure Biosystems, Dublin, CA, USA). The images were quantified using Image Studio Lite Software (Licor Inc., Lincoln, NE, USA), and actin protein (b-Actin AbCam # ab227387, 1:6000, Abcam Inc., Cambridge, UK) was used as an internal input control to normalize all measurements.

Tissue samples from the RVLM were similarly homogenized in tissue protein extraction buffer (#78510, Thermo Scientific) and were subjected to measurement of total ATP levels using an ATP detection assay kit (#700410, Cayman, Ann Arbor, MI, USA) according to the manufacturer’s instructions. Light emitted from a luciferase-mediated reaction and measured by a luminometer (Berthold Centro LB 960, Bad Wildbad, Germany) was used to calculate the measured values. Apoptotic cell death in homogenized RVLM tissues was determined following the instructed protocol of a cell death detection kit (#11544675001, Roche). This assay is based on the quantitative sandwich-enzyme immunoassay-principle using mouse monoclonal antibodies directed against DNA and histones to measure the level of cytoplasmic histone-associated DNA fragments as an index of the induced apoptotic cell death. Absorbance was measured at 405 nm using a Varioskan LUX multimode microplate reader (Thermo Scientific).

Inguinal fat tissue was homogenized with tissue protein extraction buffer (Thermo Scientific, Rockford, USA) with protease inhibitor cocktails (Roche, Mannheim, Germany). The lysate was centrifuged at 17000 rpm for 15 min and the supernatant was collected. The protein concentrations were calculated by Bradford assay solution (Bio-Rad, CA, USA). 10 μg protein was separated on 10% SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, CA, USA). The membranes were probed with the primary antibodies specific for β-actin, ATGL, HSL, LC3, ATG5, and ATG7 (Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. Secondary antibodies were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit (Santa Cruz Biotechnology, CA, USA) for 1 h at room temperature. The protein bands were detected with chem-luminescence reagents (AbClon, Seoul, Korea) by chemi-doc (Davinch-K, Seoul, Korea). β-Actin, as a loading control, was used to normalize the levels of proteins.

Each sample of dorsal skins were homogenized in tissue protein extraction buffer (Thermo scientific, Rockford, USA) with protease inhibitor cocktail tablets (Roche, Mannheim, Germany). Twenty micrograms of protein from dorsal skin was denatured with 5% SDS loading buffer. The prepared samples were loaded on 10% SDS-polyacrylamide gel electrophoresis. Then electro-transferred to activated polyvinylidene fluoride membranes. Membranes were blocked by 3% bovine serum albumin in tris-buffered saline (TBS) containing 1% tween 20 (TBS-T) and incubated with the specific antibodies at 4°C overnight (β-actin, Raf-1, p-Raf-1; Santa Cruz, CA, USA, 1:1,000 dilutions in TBS-T, ERK, p-ERK, MEK, p-MEK; Cell Signaling Technology, Danvers, MA, USA, 1:1,000 dilution in TBS-T, TrkA; Abcam, Cambridge, UK, 1:1,000 dilution in TBS-T). After washing of those membranes for 10 min three times, membranes were incubated with anti-rabbit and anti-mouse horseradish peroxidase-conjugated secondary antibody (Santa Cruz, 1:2,000 dilutions in TBS-T) for 1 h at room temperature. Then proteins were developed with an enhanced chemiluminescence detection reagent (AbClon, Seoul, Korea) by a Davinch-Western imaging system (Davinch-K, Seoul, Korea).