Phospho lrp6

The Human Phospho-Kinase Array Kit and recombinant human BMP-2/ BMP-9 were purchased from R&D Systems (Minneapolis, MN, USA). The primary antibodies against anti-human Phospho-p53 (Ser6, 9, 15, 46, 392, and Thr81), Wnt5a/b, LRP6, Phospho-LRP6, Dvl2, Dvl3, Axin1, GSK3β, Phospho-GSK3β, PI3K, Phospho-PI3K, AKT, Phospho-AKT, MDM2, and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). The iScript cDNA Synthesis Kit was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). All primers and probes (RUNX2 #Hs00231692_m1; ALPL #Hs010291444_m1; BGLAP #Hs01587814_g1; NOG #Hs00271352_s1; GAPDH #Hs99999905-m1) were purchased from Applied Biosystems, Inc. (Bedford, MA, USA). Moreover, the TaqMan MicroRNA Reverse Transcription Kit and TaqMan Universal Master mix were from Applied Biosystems, Inc.

TopFlash plasmid DNA transfection was performed using TransIt-LT1 reagent (Mirus Bio, Madison, WI, United States) according to the manufacturer’s instructions. Briefly, DNA-TransIT mix was incubated with cells for 16 h, and the transfected cells were harvested 24 h after transfection for use in the luciferase assay.
Protein lysates for Western blot analysis were prepared by using a RIPA [10 mM Tris-Cl, pH 7.2, 2 mM EDTA, 150 mM NaCl, 1% Non-idet P-40, 0.1% SDS, 50 mM NaF, 1% sodium deoxycholate, 1 mM PMSF, 1X protease inhibitor mixture set V (EMD Chemicals, Gibbstown, NJ, United States), 0.2 mM sodium vanadate] lysis buffer. Protein lysates (10 μg) were resolved on 8% polyacrylamide gel and transferred to PVDF membranes. The membranes were probed with various primary antibodies against phospho-LRP6 (1:1,000 dilution, Cell Signaling Technology), LRP6 (1:1,000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, United States), and α-tubulin (1:1,000 dilution, Santa Cruz Biotechnology). Species- and isotype-matching secondary antibodies conjugated with horseradish peroxidase (HRP) were incubated, and the HRP signal was developed using the Pierce Super Signal West Dura kit (Thermo Scientific/Pierce, Rockford, IL, United States). The signals were measured using Image J software, and each experiment was performed in duplicate.