Rabbit anti phospho yap

Embryos were fixed with 4% formaldehyde (Polysciences) for 10 min, permeabilized with 0.5% Triton X-100 (Sigma Aldrich) for 30 min, and then blocked with blocking solution (10% Fetal Bovine Serum (Hyclone), 0.1% Triton X-100) for 1 hr at room temperature, or overnight at 4°C. Primary Antibodies used were: mouse anti-CDX2 (Biogenex, CDX2-88), goat anti-SOX2 (Neuromics, GT15098), rabbit anti-PARD6B (Santa Cruz, sc-67393), rabbit anti-PARD6B (Novus Biologicals, NBP1-87337), mouse anti-PKCζ (Santa Cruz Biotechnology, sc-17781), rat anti-CDH1 (Sigma Aldrich, U3254), mouse anti-ZO1 (Thermo Fisher Scientific, 33–9100), mouse anti-YAP (Santa Cruz Biotechnology, sc101199), rabbit anti phospho-YAP (Cell Signaling Technologies, 4911), chicken anti-GFP (Aves, GFP-1020). Stains used were: Phallodin-633 (Invitrogen), DRAQ5 (Cell Signaling Technologies) and DAPI (Sigma Aldrich). Secondary antibodies conjugated to DyLight 488, Cy3 or Alexa Flour 647 fluorophores were obtained from Jackson ImmunoResearch. Embryos were imaged using an Olympus FluoView FV1000 Confocal Laser Scanning Microscope system with 20x UPlanFLN objective (0.5 NA) and 5x digital zoom. For each embryo, z-stacks were collected, with 5 µm intervals between optical sections. All embryos were imaged prior to knowledge of their genotypes.

Wild type human full length NEK1 mammalian expression plasmid was purchased from Origene (MR216282). NEK1 T141A variant was generated by site-directed mutagenesis, as previously described [23 (link)]. Generation of His-tagged N-terminal NEK1 (aa 1–480) bacterial expression plasmid was conducted as previously described. Human full length MK5 bacterial expression plasmid was purchased from Vector Builder. The following antibodies were used in this study: mouse anti-YAP (Santa Cruz Biotechnology, SCBT, Dallas, TX, USA, cat# sc101199), rabbit anti-phospho-YAP (Cell Signaling Technology, CST, Dallas, TX, USA, cat# 13008), mouse anti-NEK1 (SCBT, cat# sc 398813, Dallas, TX, USA), rabbit anti-phospho-NEK1 pT141 (lab-generated), rabbit anti-phospho-tyrosine (CST, cat# 8954S, Dallas, TX, USA), HRP-conjugated anti-β-tubulin (SCBT, Dallas, TX, USA, cat# sc-23949), mouse IgG (SCBT, Dallas, TX, USA, cat# sc-2025), and rabbit anti-actin (Abcam, Cambridge, MA, USA, cat# ab1801).

Western blot was done by running 10% SDS-PAGE, following standard blotting protocol. Primary antibodies used in this study include: rabbit anti-cleaved Caspase 3 (1:1000, Cell Signaling), rabbit anti-YAP (1:1000, Santa Cruz), rabbit anti-Phospho-YAP (1:500, Cell Signaling), rabbit anti-Lats1 (1:500, Cell Signaling), rabbit anti-Phospho-Lats1 (1:500, Cell Signaling), rabbit anti-Phospho-Mst1/2 (1:500, Cell Signaling), and mouse anti-α-Tubulin (1:2000, Sigma) antibodies. Secondary antibodies are donkey anti-rabbit or mouse IgG antibodies (Amersham).