Anti notch1val1744

λ-Phosphatase treatment,^{19 (link)} GST pull down,^{19 (link)} far western,^{19 (link)} protein extracts preparation,^{12 (link)} immunoprecipitation,^{12 (link)} immunoblotting assays^{12 (link)} and biotinylation assay^{12 (link), 62 (link)} were performed as previously described. Immunoblot analysis was performed using the following antibodies: anti-Flag (Sigma, Cat#F3165), anti-Flag-HRP (Sigma; Cat#A8592), anti-MPM2 (05-368, Millipore), anti-Notch1_Val1744 (Cell Signaling, Danvers, MA, USA, Cat#2421), anti-Notch3 (Cell Signaling; Cat#2889); anti-Notch3 M20 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-7424), anti-Pin1 (Santa Cruz Biotechnology; Cat#sc-46660), anti-β-actin (Santa Cruz Biotechnology; Cat#sc-47778), anti-Lck (Santa Cruz Biotechnology; Cat#sc-433), anti-α-tubulin (Santa Cruz Biotechnology; Cat#sc-803), anti-LaminB M20 (Santa Cruz Biotechnology; Cat#sc-6217) and anti-Hes1 (Santa Cruz Biotechnology; Cat#sc-25392). The antibody against the activated Notch3-IC protein (N3_IC-act) was kindly provided by Genentech (South San Francisco, CA, USA). The anti-N3_EC (5E1) antibody was kindly provided by Professor A Joutel.^{62 (link)} The anti-pTα antibody was kindly provided by H von Bohemer.^{63 (link)}

Samples for immunohistochemical analysis were gained from a previous study [25 (link)]. Animal experiment procedures were approved by the Provincial State Office of Western Finland with the licence ID ESAVI/3937/04.10.03/2011. Shortly, stable Pim1, Pim3, or empty plasmid (mock) overexpressing PC-3-derived prostate xenografts were allowed to grow for approximately three weeks, while part of the mice were daily treated with 50 mg/kg of the Pim inhibitor DHPCC-9. Antigen retrieval and peroxidase blocking were performed to paraffin-embedded tumor samples as previously described [9 (link)]. TBS was used instead of PBS. Samples were blocked in Dako Antibody Diluent (S0809, Agilent Technologies, Dako Denmark A/S, Glostrup, Denmark) for 10 min at RT, stained with primary antibody anti-Notch1 (Val1744, Cell Signaling Technology) 1:500 for 1 h at RT and secondary antibody Poly-HRP-Anti-rabbit IgG (DPVR55HRP, Agilent Technologies) for 30 min at RT. DAB treatment and hematoxylin counterstaining have been previously described [9 (link)]. Whole tumor scanning was performed as previously reported [25 (link)]. Double blind analysis were performed manually, and necrotic areas were left out from analysis.