Bcip nbt solution kit

Whole-mount in situ hybridization was done as previously described [28] (link). Digoxigenin (DIG)-labeled sense and antisense RNA probes were made by in vitro transcription with T7 or SP6 RNA polymerase (Roche Diagnostics GmbH, Mannheim, Germany), using templates generated by PCR. Embryos were fixed in 4% paraformaldehyde (PFA) and stored in methanol. Rehydrated embryos were sequentially treated with Proteinase K and then acetylated in acetic anhydrate solution. The pretreated samples were next hybridized at 60°C with DIG-labeled RNA probe. The probe was detected with an anti-DIG antibody-conjugated to alkaline phosphatase (Roche Diagnostics GmbH, Mannheim, Germany) and visualized by using a BCIP/NBT solution kit (Nacalai Tesque Inc.).

Cells grown in six-well plate were washed twice with PBS and solubilized with 2% CHAPS buffer (25 mM HEPES-KOH pH 7.5, 300 mM NaCl, 2% (w/v) CHAPS, protease inhibitor cocktail (Roche, Indianapolis, IN)) on ice for 30 min and then protein concentrations were determined. Proteins precipitated with TCA were lysed with SDS-PAGE sample buffer supplemented with DTT. The appropriate amounts of proteins were applied and separated on 4–12% Bis-Tris SDS-PAGE (Invitrogen) with MES or MOPS SDS running buffer (Invitrogen). After transfer, PVDF membrane were blocked and incubated with primary antibodies. Proteins were detected using alkaline phosphatase-conjugated goat anti-mouse or anti-rabbit IgG as secondary antibodies and a BCIP-NBT solution kit (Nacalai Tesque, Japan). For detecting phosphorylated ubiquitin, anti-rabbit IgG horseradish peroxidase-linked secondary antibodies (GE Healthcare Life Sciences) and Western Lightning Plus-ECL (PerkinElmer) were used.

Separated proteins were blotted on polyvinylidene difluoride membranes by semi-dry transfer. Target proteins were detected with the antibodies as follows: anti-Fra a 1e with 6× His (1:400 dilution) and alkaline phosphatase-conjugated anti-guinea pig IgG (1:3000 dilution; ab102367, Abcam, Cambridge, UK) were used to identify Fra a 1. The anti-Fra a 2 synthetic peptide (1:500 dilution) and alkaline phosphatase-conjugated anti-rabbit IgG (1:2000 dilution; S373B, Promega) were used to specifically identify Fra a 2. The BCIP-NBT Solution Kit (Nacalai Tesque) was used for alkaline phosphatase staining.