2.5 Immunoblotting
Human or rat DP cells were incubated with KY19382 for 48 hours. The
cells were washed twice with cold PBS and lysed in RIPA buffer (150 mM
NaCl; 10 mM Tris, pH 7.2; 0.1% SDS; 1% Triton X-100; 1% sodium
deoxycholate; 5 mM EDTA). Cell lysates were centrifuged at 15,920x g at
4°C for 30 minutes. The protein was separated on 8-10% SDS PAGE gels
and transferred onto PROTRAN nitrocellulose membranes (Schleicher and
Schuell Co., Keene, NH). After blocking with 5% skim milk for 1 hour at
room temperature, each membrane was blotted with the following primary
antibodies: anti-β-catenin (1:1000, Santa Cruz Technology, Dallas, TX or
1:1000, Cell Signaling, Beverly, MA), anti-α-tubulin (1:4000, Cell
Signaling or 1:2000, Oncogene Research Products, Cambridge, MA),
anti-p-GSK-3β (S9) (1:1000, Cell Signaling), anti-PCNA (1:500, Santa
Cruz Technology or 1:1000, Cell Signaling), anti-FGF9 (1:1000, Abcam,
Cambridge, MA) and anti-cytokeratin 17 (1:1000, Abcam) at
4oC overnight. Each membrane was blotted with
horse-radish peroxidase-conjugated anti-mouse (1:3000, Cell Signaling)
or anti-rabbit (1:3000, Cell Signaling) IgG secondary antibody. The
blots were visualized using enhanced chemiluminescence (Amersham
Bioscience, Buckinghamshire, UK) and a luminescent image analyzer
(LAS-4000; Fujifilm, Tokyo, Japan).