Kpl block

Embryos were collected at various stages (Nieuwkoop and Faber, 1994 ) for lysate creation. Protein lysates from 20 pooled embryos of the same stage were created as described previously (Kim et al., 2002 (link)), and one embryo equivalent was added per lane of an 8% SDS-PAGE polyacrylamide gel. Following transblotting of the protein onto a 0.45 μm PVDF membrane (Thermo Scientific), the blot was blocked for 3 h in KPL block (SeraCare) at room temperature. After blocking, the membrane was incubated overnight at 4°C in 1:500 mouse anti-Dnmbp antibody (Abcam 88534) or 1:1000 rabbit anti-GAPDH antibody (Santa Cruz FL-335). Blots were rinsed with TBST and incubated in 1:5000 goat anti-mouse or goat anti-rabbit IgG horseradish peroxidase secondary antibody (BioRad, Hercules, CA) for 2 h at room temperature. Blots were rinsed again in TBST and imaged using enhanced chemiluminescence (Pierce Supersignal West Pico) on a BioRad ChemiDoc XRS+.

Cells were trypsinized from plates, collected and washed twice in PBS prior to being resuspended in 2X Laemmli (Biorad) plus 100 μm dithiothreitol. The resulting cell lysates were then boiled at 95°C for 30 minutes. Lysates were run on an 8% SDS-PAGE gel and the protein was then transblotted onto a 0.2 μm nitrocellulose membrane (GE Healthcare), followed by blocking for 3 hours in KPL block (SeraCare) at room temperature. Blots were incubated in 1:1000 rabbit anti-GAPDH (Santa Cruz sc-25778), 1:1000 rabbit anti-Daam1 (Proteintech 14876-1-AP) or 1:1000 rabbit anti-β-catenin [65 (link)] primary antibodies for 1–2 hours. Blots were then washed with TBST, incubated in goat anti-rabbit IgG horseradish peroxidase secondary antibody (1:5000, BioRad) for 2 hours at room temperature and then washed again with TBST prior to imaging on a BioRad ChemiDoc XRS+ imaging system using enhanced chemiluminescence (Pierce Supersignal West Pico). Preparation of MDCK cell extracts and Western blot analysis presented in Fig 2 were carried out following the protocol described by [55 (link)].