Anti ck1α

Western blot, immunofluorescence (IF),⁵¹ (link) and immunohistochemistry (IHC)⁵² (link) were performed as previously described. Anti-Notch1, anti-β-catenin, anti-p84, anti-APC, anti-GSK3β, anti-CK1α, and anti-β-TrCP antibodies were purchased from Abcam. Anti-Nanog, anti-BMI1, anti-Oct4, and anti-HES1 antibodies were obtained from Cell Signaling Technologies. Anti-TET3 and anti-actin antibodies were purchased from GeneTex and Sigma, respectively. Western band intensity was quantified using ImageJ software (NIH).

Lysates of cells or tumor tissues were prepared with lysis buffer containing 20 mM Tris⋅HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerol phosphate, 1 mM sodium orthovanadate, 2 µg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride. Equal amounts of proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes. Immunoblotting was performed with anti-phosphorylated LRP6 (Ser1490) (2568; Cell Signaling), anti-LRP6 (2560; Cell Signaling), DVL2 (3216; Cell Signaling), anti-active β-catenin (8814; Cell Signaling), anti-β-catenin (sc-7963; Santa Cruz), anti-CK1α (108296; Abcam), anti-CK1δ (sc-55554; Santa Cruz), anti-CK1ε (12448; Cell Signaling), anti-CD44 (3570; Cell Signaling), anti-Slug (9585; Cell Signaling), anti-Snail (3879; Cell Signaling), anti-GAPDH (HC-301; TransGen Biotech), and anti-β-actin (HC-201; TransGen Biotech).