Bcip nbt kit

Cells were lysed, and proteins were separated by SDS-PAGE and transferred to PVDF membrane. After blocking of non-specific binding, immunoblots were incubated with monoclonal antibodies against the N-terminal region (a.a. 1–181) of WT1 protein (6F-H2, Dako Cytomation, Carpinteria, CA), GAPDH (6C5, Millipore, Temecula, CA) and His-tag (Anti-Xpress antibody, Invitrogen, Carlsbad, CA) or polyclonal antibody against the C-terminal region (a.a. 431–450) of WT1 protein (C-19, Santa Cruz Biotechnology), followed by incubation with an anti-mouse or rabbit IgG antibody conjugated with alkaline phosphatase (Santa Cruz Biotechnology), and visualized using BCIP/NBT kit (Nacalai Tesque, Kyoto, Japan).

Proteins were separated by SDS-PAGE and transferred to Immobilon polyvinylidene difluoride membrane. After blocking of non-specific binding, the membranes were incubated with the first antibodies, followed by incubation with the corresponding secondary antibodies conjugated with alkaline phosphatase, and visualized using BCIP/NBT kit (Nacalai Tesque, Kyoto, Japan). Polyclonal anti-EF2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-GAPDH (Chemicon International, Temecula, CA, USA) were used as the first antibodies.