Horseradish peroxidase hrp conjugated goat anti mouse rabbit igg

Two days after transfection, cell lysates were clarified by centrifugation at 10,000 × g for 20 min at 4 °C. Samples were incubated with 1 μg of indicated antibody and 20 μl of 50% (v/v) Protein A-agarose (Pierce) for 2 h with gentle rocking. After three washes with lysis buffer, precipitated complexes were solubilized by boiling in Laemmli sample buffer, fractionated by SDS-PAGE, and transferred onto polyvinylidene fluoride (PVDF) membranes, which were blocked with phosphate buffered saline (PBS; pH 7.5) containing 0.1% gelatin and 0.05% Tween 20 and blotted with the indicated antibodies. After two washes, blots were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse/rabbit IgG (Jackson ImmunoResearch Laboratories) for 1 h. After washing, reactive bands were visualized using the VisGlow chemiluminescent substrate, HRP system (Visual Protein, Taipei). For lectin blot analysis, membranes were blocked with PBS (pH 7.5) containing 0.1% gelatin and 0.05% Tween 20, detected with biotinylated Lens culinaris lectin (LCA) (Vector Laboratories, Burlingame, CA, USA), then incubated with HRP-conjugated streptavidin (Vector Laboratories).

In total, 5 × 10⁵ fresh CD34⁺ cells were cultured with JNK-IN-8 (2 μM) or DMSO (v/v 0.01%) supplemented with 10% serum for 30 min, and then were pelleted and washed with PBS. The pellets were resuspended with 45 μl PBS and 15 μl lysis buffer (200 mM Tris-HCL, 8% SDS, 400 mM DTT, 0.1% bromophenol blue, 40% glycerol) and incubated at 100 °C for 10 min for lysis. The lysed solutions were electrophoresed in 10% SDS-PAGE and transferred onto PVDF membrane. The membranes were incubated with the appropriate primary antibodies (rabbit anti-total-c-Jun, Cell signaling, Cat:9165; rabbit anti-phosphor-c-Jun (ser63), Cell signaling, Cat: 9261; mouse anti-GAPDH, CWBIO, Cat: 0100 A) in 4% nonfat milk at 4 °C overnight. Then they were incubated with the horseradish peroxidase (HRP)-conjugated goat anti-mouse/rabbit IgG (Jackson Laboratories) as secondary antibodies and visualized by Tanon infrared imaging system.