For Western blot, preparation of lysate from cell culture was described as previously reported.
As the cloned miRNAs were derived from protonema and gametophores, total RNA from these tissues was used for RNA gel blot preparation.
For Northern blot preparation, 20 μg of total RNA was separated by 1% formaldehyde-agarose gel electrophoresis and transferred to nylon membrane (Hybond N, Amersham Pharmacia Biotech, Pollards Wood, UK) by standard methods.
For quantitative Southern dot-blot, preparations of serial dilutions of yeast genomic DNAs were denatured in 0.4 M NaOH, spotted 2 μL per dot onto dry nylon membrane (Hybond N+, Amersham Pharmacia Biotech).
BL participated in GFP assay, GUS analysis, Western blot, figures preparation and manuscript revising.
A total of 20 µg purified protein was separated in a preparative gel, followed by blotting and preparation of membrane strips (each containing 1.2 1.4 µg protein).
MHW carried out blotting, culture preparation, Wnt-1 cell cloning, proliferation assay.
Western blotting and preparation of conditioned medium and cell extracts were performed according to the procedure described previously [ 26].
Compared to direct TH blotting, NaPTA preparations often revealed a slower electrophoretic migration of up to 0.5 kDa (Fig. 3C, 3D and data not shown), as previously reported [14].
For Western blotting, cell lysate preparation, gel electrophoresis and blotting were performed as described previously [11] using antibodies for ERG (sc-353, Santa Cruz Biotechnology Inc., Heidelberg, Germany), ESE-3 (ETS3 Clone 5A5, Lab Vision, Fremont CA. USA), EZH2 (612667 BD Biosciences, San Jose, USA), Nkx3.1 (SC-15022,Santa Cruz Biotechnology Inc).
Western blotting, total RNA preparation and real-time PCR For western blotting, nuclear extracts were separated by SDS-PAGE and blotted on nitrocellulose membranes as described [ 13].
