Microchemi 4.2 system

The total protein was extracted from each intervention group by lysing the cells in a radioimmunoprecipitation assay buffer (Beyotime, Shanghai, China). Quantitative analysis of the protein concentrations was performed with a special analytical kit (Beyotime, Shanghai, China). The same amount of proteins was obtained by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were then blocked with 5% skimmed milk powder in TBST (Tris-buffered saline solution containing 0.1% Tween 20) and cultured with primary and secondary antibodies. The specific protocol of the western blotting is based on the literature [23 (link)]. After washing, the staining was visualised with an enhanced chemiluminescence kit (Beyotime, Shanghai, China), and the specific strip was measured with the MicroChemi 4.2 system (DNR Bio-Imaging Systems, Jerusalem, Israel).

Semiquantitative immunodetection of serum cytokines, chemokines, growth factors, angiogenesis markers, and other soluble proteins was performed using the immuno-dot-blot method in the Proteome Profiler Human XL Cytokine Array Kit (ARY022B, R&D Systems, Inc., Abingdon, UK). A number of 105 captured antibodies, along with reference controls, were spotted in duplicate on nitrocellulose membranes and incubated overnight with 100 mL of pooled serum samples. Each of the four pools was obtained by mixing the serum samples from CKD patients in stages 4, 3, and 2, respectively; the 4th pool was assigned to control sera. The protocol recommended by the manufacturer was followed accordingly. The membranes were incubated with biotinylated detection antibodies, streptavidin-horseradish peroxidase (HRP), and chemoluminescent detection reagents. Chemiluminescence signals, corresponding to the amount of protein bound, were detected using the MicroChemi 4.2 System (DNR Bio-Imaging Systems, Israel), and the intensity of the chemiluminescence signals (pixel densities) was measured using ImageJ 1.42 software (National Institute of Health, Bethesda, MD, USA). For each measured analyte, the average signal of the duplicate spots was determined and normalised to the average signal of the reference spots after being corrected with the background signal.

Total proteins were extracted using PRO-PREP buffer (iNtRON Biotechnology Inc., Seoul, Korea) containing proteinase inhibitors and phosphatase inhibitors (GenDEPOT, Barker, TX). Homogenates were centrifuged at 13,000 rpm for 15 min at 4 °C, supernatant were collected, and protein concentration was determined using the BCA protein assay kit (Thermo Scientific, Rockford, IL). Equal amounts of protein were resolved on SDS-PAGE gels, and then transferred to PVDF membranes. Primary antibodies against the following proteins were used: PGC1α (Abcam, Cambridge, UK), Troponin I-SS (C-19), Troponin I-FS (G-7) (Santa Cruz Biotechnology, CA, USA), and GAPDH (Cell Signaling Technology, MA, USA). The membranes were then incubated with anti-rabbit or anti-mouse IgG horse-radish peroxidase-linked secondary antibody (AbClon, Korea), and then visualized with Micro-Chemi 4.2 system (DNR Bio Imaging Systems, Israel). The target protein levels were then normalized against the GAPDH protein levels. Band intensities were measured with image J software (NIH, USA).

As a first testing approach, we used Proteome Profiler Human XL Cytokine Array Kit (ARY022B, R&D Systems, Abingdon, UK) for semiquantitative determination of cytokines, chemokines, growth factors, angiogenesis markers and other soluble proteins in plasma samples of MPN patients and controls. The multianalyte assay contains nitrocellulose membranes, each being spotted in duplicate with capture antibodies to 105 distinct analytes and also with control antibodies. The protocol recommended by the manufacturer was followed. Briefly, each membrane was incubated with 100 μL of undiluted plasma sample at 4°C overnight. After extensive washing to remove unbound materials, the membranes were incubated with biotinylated detection antibodies. Subsequently, streptavidin‐HRP was added followed by chemiluminescent detection reagents. The chemiluminescent signals generated at each spot corresponding to the amount of protein bound were recorded by the MicroChemi 4.2 system (DNR Bio‐Imaging Systems, Israel) after membrane exposure. The intensity of signals (pixel densities) was quantified with ImageJ 1.42 (National Institute of Health, Bethesda, MD, USA). For each analyte, the average signal of the duplicate spots was calculated, corrected for background signal and normalized to the average signal of the membrane reference spots.

Total proteins were extracted using PRO-PREP buffer (iNtRON Biotechnology Inc., Seoul, Korea) containing phosphatase inhibitors (GenDEPOT, Barker, TX, USA), separated by SDS-PAGE, transferred to PVDF membranes (Millipore, Billerica, MA, USA), and analysed by immunoblotting. Primary antibodies against the following proteins were used: UCP1, PGC1α, CPT2, PPARα, AHNAK, and OXPHOS (oxidative phosphorylation) complexes (all from Abcam); pHSL, HSL, pPKA substrates, and GAPDH (all from Cell Signaling Technology, Beverly, MA, USA); and tyrosine hydroxylase (Millipore). The antibodies were diluted 1:1,000 with TBS containing 0.1% (v/v) Tween-20 (TBST, Biosesang, Seongnam, Korea). The membranes were then incubated with a peroxidase-conjugated secondary antibody (AbClon, Seoul, Korea), and antibody-specific signals were detected by enhanced chemiluminescence and quantified using the MicroChemi 4.2 system (DNR Bio Imaging Systems, Jerusalem, Israel).

Frozen lung tissues were sonicated in RIPA lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 1 mM EDTA) containing protease inhibitor cocktail (Roche Applied Bioscience). Protein content was determined by Bradford assay (Amresco) staining using bovine serum albumin as a standard. Equal amounts of protein were loaded on 10%, 12%, or 15% Tris-glycine gels for electrophoresis. Proteins were wet-transferred to PVDF membranes (Millipore) and then probed with the indicated antibodies. The primary antibodies and dilutions used were as follows: anti-surfactant protein B rabbit polyclonal antibody (1:3,000, #07-614, Millipore), anti-surfactant protein C rabbit polyclonal antibody (1:1000, #AB3786, Millipore), anti-surfactant protein A rabbit polyclonal antibody (1:1000, #AB3420, Millipore), anti-p62 rabbit polyclonal antibody (1:1000, #P0067, Sigma), LC3B rabbit polyclonal antibody (1:1000, #12741, Cell Signaling Technology), anti-ATG7 rabbit polyclonal antibody (1:1000, #A2856, Sigma) and anti-actin mouse polyclonal antibody (1:1000, #TA-09, Zsgb-bio, China). Immunoreactivity was detected using horseradish peroxidase-conjugated secondary antibodies. Chemiluminescence substrates were used (Tiangen, Beijing, China). The images were captured using a MicroChemi 4.2 system (DNR Bio Imaging Systems, Jerusalem, Israel).

For immunoblot analysis, cells were lysed in PRO-PREP buffer (iNtRON Biotechnology Inc., Seoul, Korea) containing a phosphatase-inhibitor cocktail (GenDEPOT, Barker, TX, USA). Protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, Massachusetts, USA), and subjected to immunoblot analysis. Cytosolic and nuclear fractions used in immunoblot analysis were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, Rockford, IL, USA), according to the manufacturer's instructions. For immunoprecipitation experiments, lysates were immunoprecipitated with an anti-HA antibody at 4°C overnight. Immunocomplexes were washed three times with lysis buffer (1% Triton X-100, 20 mM HEPES at pH 7.5, 150 mM NaCl, 12.5 mM, 10% glycerol, 5 mM EDTA, proteinase inhibitor cocktail [GenDEPOT], and a phosphatase inhibitor), mixed with 2X sample buffer, and separated from the protein A/G agarose beads (Santa Cruz) by boiling. Immunoblot analysis was performed using the indicated antibodies. Proteins were visualized by ECL chemiluminescence (AbClon, Seoul, Korea). GAPDH was detected as a loading control. Immunoreactive signals were detected through their enhanced chemiluminescence and recorded using the MicroChemi 4.2 system (DNR Bio-Imaging Systems, Jerusalem, Israel).