Enhanced chemiluminescence technique

Cultured Cells were washed once with PBS buffer then lysed in lysis buffer (1% SDS, 50 mM Tris, pH 7.4, 0.15 M NaCl, 1 mM NaF, 10 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 mM EDTA) for 5 min and passed through a 27-gauge needle. Lysates were centrifuged at 12,000 g for 1 min and protein concentrations were determined using a Bio-Rad DC protein assay. Equal amounts of protein were separated by 4∼20% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% skim milk or 3% bovine serum albumin in TBST (10 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween 20) for 1 h. Primary and secondary antibodies were used according to the manufacturer's instructions, and this was followed by detection with an enhanced chemiluminescence technique (Amersham Biosciences). Western quantitations were performed as follows: Western blots were scanned by a densitometer to obtain the density of each band. Then, the ratio of the density of a band of interest to that of its internal control band (GAPDH) was relative to the ratio from the negative control experiments. Each Western blotting was repeated at least twice.

Cells were seeded at 60%–80% confluence at transfection. Within 24 h, individual siRNAs including control siRNA, LRP5 siRNA, and LRP6 siRNA (Ambion) were transfected using lipofectamin RNAiMAX reagent (Invitrogen) according to the manufacturer’s instruction. Western blot analysis of cell lysates was performed as described previously.^{45 (link),46 (link)} The antibodies used were anti- LRP6 [EPR2423(2)] (Abcam, ab134146, 1:500), anti-β-actin (Cell Signaling, 3700, 1:1 000), anti-pCREB (Abcam, ab32096, 1:5 000), anti-CREB (Cell Signaling, 9197, 1:1 000), anti-pSmad2/3 (Cell Signaling, 8828, 1:1 000), anti-Smad2/3 (Cell Signaling, 3102, 1:1 000), anti-phospho-Smad1/5/8 (MilliporeSigma, AB3848, 1:1 000), anti-β-catenin (cell signaling, 9562, 1:1 000), anti-Na-K-ATPase (Abcam, ab7671, 1:1 000), and anti-Flag (Sigma-Aldrich, F3165, 1:2 000). All blots were developed by the enhanced chemiluminescence technique (Amersham). For intracellular cAMP measurement, an enzyme immunoassay kit (Cayman Chemical) was used, and the sample preparation and cAMP concentration detection were performed according to the manufacturer’s instruction.