Mouse anti e cadherin

The following primary antibodies were employed for Western blotting: mouse anti-β-tubulin (1:1000, cod. T5201; Sigma-Aldrich Corp.), mouse anti-β-actin (1:2000, cod. A5441; Sigma-Aldrich Corp.), rabbit anti-GAPDH (1:1000, cod. G9545, Sigma-Aldrich Corp.), mouse anti-cathepsin D (1:100, cod. IM03; Calbiochem, St. Louis, MO, USA), mouse anti-histone H3 (1:500, cod. 61475; Active Motif, Carlsbad, CA, USA). The secondary antibodies used for Western blot analysis were the following: horseradish peroxidase-conjugated goat anti-mouse IgG (1:10,000, cod. 170-6516; Bio-Rad, Hercules, CA, USA) and horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000, cod. 170-6515: Bio-Rad, Hercules, CA, USA). The primary antibodies employed for immunofluorescence staining are listed below: mouse anti-cathepsin D (1:100, cod. IM03; Calbiochem), rabbit anti-cathepsin D (1:100; EMD Biosciences, Calbiochem, San Diego, CA, USA), rabbit anti-p27 (1:100, cod. 2552; Cell Signaling, Danvers, MA, USA), rabbit anti-Ki-67 (1:100, cod. HPA001164; Sigma-Aldrich), mouse anti-E-cadherin (1:50, cod. 14472S; Cell Signaling) and rabbit anti-N-cadherin (1:50, cod. 4061S; Cell Signaling). The secondary antibodies were goat-anti rabbit IgG Alexa Fluor Plus 488 (1:1000, cod. A32731; Invitrogen, Waltham, MA, USA) and goat-anti mouse IgG Alexa Fluor Plus 555 (1:1000, cod. A32727; Invitrogen).

Extracts from E18.5 and postnatal day 0 (P0) mice or transfected cells were incubated on ice for 30 min in cold RIPA buffer (50 mm Tris-HCl, 150 mm NaCl, 1 mm EDTA, 1% NP-40) with PMSF, phosphatase inhibitors and protease inhibitors. Afterwards the lysates were preincubated with Ryk or Vangl2 antibody for 1 h. Then antibodies were immobilized on Protein A/G agarose beads (Santa Cruz Biotechnology) overnight at 4 °C. The beads were precipitated by centrifugation at 6 000 r.p.m. for 1 min, and immunoprecipitates were washed three times in RIPA buffer and subjected to western blotting analysis. The protein samples were separated by 10% SDS-PAGE and transferred onto PVDF membranes. After blocking in blocking buffer (0.5% milk) for 1 h at room temperature, the blots were incubated with rabbit anti-Ryk (1:500), goat anti-Vangl2 (1:500), rabbit anti-Fzd3 (1:500), mouse anti-Dvl1 (1:200), rabbit anti-GAPDH (1:1000; Sigma-Aldrich), mouse anti-E-cadherin (1:1000), mouse anti-FLAG (1:1000) or rabbit anti-HA (1:1000), and subsequently with a peroxidase-conjugated secondary antibody.

Cells were solubilized in cold RIPA lysis buffer. Proteins were separated with 12% SDS-PAGE, and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with TBST containing 5% skimmed milk at 37°C for 2 hours. Then, the membrane was incubated with mouse anti-SALL4 (1:100; Sigma Aldrich), mouse anti-N-cadherin (1:100; Sigma Aldrich), mouse anti-E-cadherin (1:400; Sigma Aldrich), and mouse anti-GAPDH primary antibodies (1:200; Sigma Aldrich), respectively, at room temperature for 2 hours. After being washed by PBST for 4 times with 10 minutes each time, the membrane was incubated with the goat anti-mouse secondary antibodies (1:5000; Sigma Aldrich) at 4°C overnight. After being washed by PBST for 4 times with 10 minutes each time, ECL kit (Pierce Chemical, Rockford, IL, USA) was used to perform chemiluminent detection. Image-Pro plus software 6.0 was used to analyze the relative protein expression, represented as the density ratio versus GAPDH.