Immobilon western hrp detection reagent

Whole proteins from mouse aortas and harvested cells were extracted using radioimmunoprecipitation assay (RIPA) buffer. Cytoplasmic extracts of HUVECs were extracted using a hypotonic buffer (1 M HEPES, pH 7.9, 1 M MgCl₂, 2.5 M KCl, 1 M DTT, and protease inhibitors). Mouse aortic tissue extracts (IL-6, IL-1β, and KLF2), total cell extracts (eNOS, KLF2, IL-1β, and VCAM-1), and cytoplasmic extracts (ERK, phospho-ERK, JNK, phospho-JNK, P38, and phospho-p38) were fractionated by electrophoresis on 10% SDS-PAGE gels, and transferred onto nitrocellulose membranes. The membranes were blotted with antibodies specific to each protein. The peroxidases bound to the blots were detected with the Immobilon Western HRP detection reagent (Millipore, Billerica, MA) using an Image Reader (LAS-3000 Imaging System; Fuji Photo Film, Tokyo, Japan).

The brain tissues of each rat were extracted by cytoplasmic extracts (for IκBα and β-actin), and nuclear extracts (for NF-κB) fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membranes. Cytosolic extracts were prepared in hypotonic buffer 10 mM N-(2-hydroxyethyl)-piperazine-N-2-ethanesulfonic acid (HEPES, pH 7.6), 10 mM KCl, 2 mM MgCl₂, 1 mM dithiothreitol, 0.1 mM ethylene diamine tetra acetic acid (EDTA), and 0.1 mM phenyl methyl sulfonyl fluoride (PMSF). Nuclear extracts were prepared in hypertonic buffer consisting of 50 mM HEPES (pH 7.9), 400 mM KCl, 0.1 mM EDTA, and 10% glycerol. Each transfer membrane was blocked overnight at 4°C with blocking solution [10 mM Tris–HCl (pH 7.4), 125 mM NaCl, 0.1% Tween 20, and 5% skim milk] incubated with specific antibodies for 1–3 h at room temperature. The blots were washed 3 times using washing buffer (20 mM Tris, 160 mM NaCl, and 0.1% Tween 20), followed by incubation for 1 hour with the appropriate horseradish peroxidase- conjugated secondary antibody. The peroxidase bound to the blot was detected using the Immobilon Western HRP detection reagent (Millipore, Billerica, MA, U.S.A.). The ratio of the protein interested was subjected to β-actin and was densitometrically analyzed by Image J software (NIH, Bethsda, MD).

Hypothalamus of brain tissues were homogenized in ice-cold RIPA buffer supplemented with protease (Calbiochem, San Diego, CA, USA) and phosphatase (Sigma-Aldrich, St. Louis, MO, USA) inhibitors. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay (Sigma-Aldrich). Equal amounts of protein extracts (50 µg) were fractionated by SDS-PAGE and transferred to 0.45-µm nitrocellulose membranes. Membrane blocking was performed by incubating for 1 h at room temperature with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween-20. Then, the membranes were incubated overnight at 4°C with the POMC and actin primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a 1∶1000 dilution. The blots were washed three times with washing buffer (20 mM Tris, 160 mM NaCl and 0.1% Tween 20), followed by a 1-h incubation with the appropriate horseradish peroxidase-conjugated secondary antibody. The peroxidase activity was detected using the Immobilon Western HRP detection reagent (Millipore, Billerica, MA, USA) using an Image Reader (Thermo Fisher Scientific, Pittsburgh, PA, USA).