Anti phosphorylated lrp6

A Western blot was performed as described previously [45 (link)]. AMOC2 cells were starved in FBS free medium for 12 hours, then added Wnt3a, DKK1, Wnt5a and MMP10 at concentrations 40 ng/mL, 500 ng/mL, 100 ng/mL and 10 U, respectively and incubated for 2 hours. The cells were lysed by an SDS sample buffer. Protein samples were applied to SDS-PAGE, and separated proteins were transferred onto a PVDF membrane (Immobion-P transfer membrane, Melck). After blocking with 5% skim milk in Tris-buffered saline containing 0.03% Tween20 (TBS-T) for 40 min, the membrane was incubated with a primary antibody at a dilution of 1:1000 in TBS-T containing 5% skim milk for 40 min at room temperature. Anti-LRP6 (Cell Signaling) and anti-phosphorylated LRP6 (Cell Signaling) antibodies were used.

Western blot analysis detected the phosphorylated level of LRP6. Mouse retina was dissected and homogenized for protein extraction. Equal amounts (50 μg) of total protein from each sample were resolved by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. The membrane was blocked with 5 % nonfat milk and separately blotted with primary antibodies (anti-Non-phosphorylated β-Catenin, Cell Signaling Technology; anti-phosphorylated LRP6, Cell Signaling Technology; and anti-LRP6, self-made). After thorough washes, the membrane was incubated with the peroxidase-conjugated horse anti-rabbit or horse anti-mouse antibody (Vector Lab). The signal was developed with the enhanced chemiluminescence (ECL) system (Pierce, Rockford, IL, USA). Membranes were stripped and re-blotted with anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA) as the loading control. Densitometry of the signal bands on digital images was quantified using FluorChem Q software (ProteinSimple, Santa Clara, CA, USA).