Ab32096

Proteins were extracted from HFLs using RIPA lysis buffer (Beyotime, P0013B) and quantified by the BCA protein assay kit (Beyotime, P0012S). After separation by 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis, the proteins were transferred onto a Hybond-P membrane (Amersham Biosciences). The membrane was blocked with 8% skim milk powder in TBST for 1 h at 25 °C, and then incubated with rabbit anti-human pCRTC1 antibody (Sigma, ABE560, dilution 1:1000), rabbit anti-human pCRTC2 antibody (Abcam, ab109081, dilution 1: 1000), rabbit anti-human CRTC2 antibody (Cell Signaling Technology, 9198, dilution 1: 1000), rabbit anti-human pCREB antibody (Abcam, ab32096, dilution 1: 1000) and GADPH (Cell Signaling Technology, 5174, dilution 1: 2000) at 4 °C for 12 h. After extensive washing with 1% TBST, the membrane was incubated with the horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Cell Signaling Technology, 7074, dilution 1: 5000) for 1 h at room temperature. Bands were visualized with an enhanced chemiluminescence detection kit (Thermo, WP20005). Quantitative analyses were performed using Image J software, with GADPH as the internal standard [15 ]. Images of full-length western blots presented in supporting information.

Cellular lysates from cultured neurons were prepared using RIPA buffer (ThermoFisher Scientific) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (ThermoFisher Scientific). After measuring protein concentration with the BCA protein assay kit (Pierce, Rockford, IL), protein samples were loaded to 10% SDS-polyacrylamide gels. After separation, proteins were transferred onto polyvinylidene difloride (PVDF) membranes. The PVDF membranes were blocked with 5% bovine serum albumin and then probed with primary antibodies (phospho-ERK1/2, Abcam, ab76299, 1:5000 dilution; ERK1/2, Abcam, ab184699, 1:5000 dilution; phospho-CREB, Abcam, ab32096, 1:5000 dilution; CREB, Cell Signaling Technology, 9197, 1:1000 dilution) at 4 °C overnight. Membranes were then incubated with peroxidase-conjugated secondary antibody for 1 h; detection was performed using West Pico PLUS chemiluminescent substrate (ThermoFisher Scientific) and scanned with an Amersham Image 600 imager. Densitometry analysis was done with ImageJ.

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.