Horseradish peroxidase conjugated secondary antibody

Protein extracts were obtained as described in protein extracts paragraph with a complete lysis buffer.
10 µg of proteins were mixed with Laemmli buffer and then denatured at 95 °C for 5 min. Proteins were separated in a 13% acrylamide SDS-PAGE and, after electrophoresis, transferred to nitrocellulose filters for western blot analysis.
Membranes blocking, washing and antibody incubation were performed according to manufacturer’s instructions. Antibodies against β-tubulin (1:2000, Sigma-Aldrich, USA) and GTP-tubulin (1:1000, Adipogen, USA) were used. After incubation with primary antibodies, membranes were washed and then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1:2000) (anti-mouse, Chemicon, USA; anti-rabbit, PerkinElmer, USA; anti-human, Sigma-Aldrich, USA). Immunoreactive proteins were visualized using an ECL chemiluminescence system (Amersham, USA).

Proteins (50 μg) were resolved by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, MA, USA). The membrane was first incubated with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies (Chemicon International). Signals were visualized using enhanced chemiluminescence detection reagent from Millipore, and the images were obtained using a Luminescence/Fluorescence Imaging System (LAS-4000; Fuji).
For immunoprecipitation, cell lysates containing 500 μg of protein were pre-cleared by protein A/G Sepharose beads (Millipore) and then incubated with anti-L-FABP or anti-VEGFR2 antibody overnight at 4°C. The immune complexes were washed three times in ice-cold PBS and subsequently captured by protein A/G Sepharose beads, and then the immunoprecipitated proteins were subjected to western blot analysis.

For immunoblotting, we homogenized tissues in RIPA buffer (Wako Pure Chemical Industries, Ltd., Tokyo) containing protease inhibitors (Takara Bio Inc., Shiga, Japan) and a phosphatase inhibitor (Nacalai Tesque, Kyoto, Japan) and collected the supernatants. Proteins (5 μg/lane) were separated on 5–20% gradient SDS–polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (GE Healthcare Bio Sciences, Piscataway, NJ). After blocking with TBS-T buffer containing either 3% bovine serum albumin or 5% skim milk, the membranes were incubated with antibodies against phospho-eNOS (Ser¹¹⁷⁷, 1:1000, Cell Signaling Technology, Beverly, MA), phospho-eNOS (Thr⁴⁹⁵, 1:1000, Cell Signaling Technology) and total eNOS (1:1000, BD Biosciences, San Diego, CA) or a peroxidase-conjugated antibody against β-actin (1:50000, Sigma–Aldrich, St. Louis, MO) [29 (link)] and antibody binding was detected with horseradish peroxidase-conjugated secondary antibodies (1:2000; Chemicon) using an enhanced chemiluminescence system (GE Healthcare Japan, Tokyo). The band intensity of phospho-eNOS (Ser¹¹⁷⁷ and Thr⁴⁹⁵) was scanned in gray scale at the maximum resolution of at least 600 dpi using NIH Image J 1.47 and arbitrary ratio normalized to the band intensity of total-eNOS were used.