Rabbit anti pkd1

The primary antibodies used were rabbit anti-E-cadherin, rabbit anti-N-cadherin, mouse anti-β-catenin and rabbit anti-PKD1 (1/500 for western blot and 1/50 for immunofluorescence; Santa Cruz Biotechnology, Santa Cruz, CA), goat anti-actin (1/200 for western blot; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-cyclin D1 (1/1000 for western blot, Cell signaling technology, Denvers, MA). Horseradish peroxidase-conjugated secondary antibodies used were goat anti-rabbit IgG (1/2000; Dako, Glostrup, Denmark), rabbit anti-goat IgG (1/2000 Santa Cruz Biotechnology) and goat anti-mouse IgG (1/5000; Rockland, Gilbertsville, PA). The Alexa Fluor-conjugated secondary antibodies were Alexa-Fluor-594-conjugated donkey anti-rabbit IgG, Alexa-Fluor-488-conjugated donkey anti-mouse IgG conjugated (1/200; Invitrogen, Cergy-Pontoise, France). Actin was stained with Alexa-Fluor-488-conjugated phalloidin (1/50; Invitrogen, Cergy-Pontoise, France). The nucleus was stained with 4',6-diamidino-2-phenylindole DAPI (1/50000; Invitrogen, Cergy-Pontoise, France). Gö6976 and Gö6983 were purchased from Calbiochem (Darmstadt, Germany). All other biochemicals were from Sigma-Aldrich (St. Louis, MO).

Cells at different time points of differentiation were fixed using 4% paraformaldehyde (PFA). Samples were then subjected to treatment with NH₄Cl and blocking with 0.3% TritonX-containing BSA before incubation with the primary antibodies. Mouse anti SSEA-1 (Santa Cruz Biotechnology, USA), 1:200, over-night at 4 °C; rabbit anti-PKD1 (Santa Cruz, USA), 1:200, 2 h at room temperature (RT); rabbit anti-PKD2 (Orbigen, USA), 1:1000, 1 h at RT; mouse anti-Oct3/4 (Santa Cruz, USA), 1:200, overnight at 4 °C; rat anti-CD31, (Becton Dickinson, USA), 1:100, 1 h RT; mouse anti α-Actin (Sigma Aldrich, Germany) 1:100, 1 h 37 °C. Samples were further incubated with fluorescence labelled secondary antibodies Alexa Fluor® 488 (green), Alexa Fluor® 568 (red), Alexa Fluor® 647 (magenta) (Life-technologies, all diluted 1:500). For germ layer-specific staining we used the chicken anti β–tubulin-III antibody ((TUBB3), Millipore, Billerica, MA), 1:1000, goat anti-human Brachyury (R&D Systems, Minneapolis, MN, USA, www.rndsystems.com), 1:100, o.N. 4°, AF2085 and goat anti-human SOX17 (R&D Systems), 1:500, o.N. 4°, AF1924. Nuclei were stained with DAPI (blue) (1:20,000). Images were captured using an upright fluorescence Zeiss Axioimager Z1 microscope and analysed using Axiovision software (Zeiss, Germany).