Kinase screening library

BODIPY™ 558/568 C12 (4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid, Thermo Fischer Scientific), Hoechst (Sigma), sodium arsenite (Fischer Chemical), cycloheximide (Sigma), BODIPY™ 493/503 (ThermoFischer Scientific, D3922), Rosiglitazone (Sigma), Clofibrate (Sigma), GW501516 (Sigma), 2-bromopalmitic acid (Sigma), streptavidin-HRP (Thermo Scientific), fatty acid-free BSA (PAN Biotech), DMEM (PAN Biotech), FBS (PAN Biotech), PBS (PAN Biotech), methanol (Roth), chlorophorm (Sigma), aprotinin (Roth), leupeptin (Roth), phenylmethylsulfonyl fluoride (PMSF, Sigma), Fasnall (Sigma), and kinase screening library (10505, Cayman Chemical).

Human ZNRF3 was analyzed with the kinase prediction online tool, GPS 3.0 (http://gps.biocuckoo.cn/index.php, Wuhan, China) to select candidates for 4Y kinase screening. Additional PTK candidates were selected based on plasma membrane or cortical localization (Shuai and Liu, 2003 (link); Ingley, 2008 (link); Mocsai et al., 2010 (link)). For screening, TetOn ZNRF3-HA H1703 cells were induced with doxycycline (DOX) (200 ng/ml). After 24 hr, tyrosine kinase inhibitors (500 nM; stocks in DMSO) from a Kinase screening library (Cayman, Ann Arbor, MI) were added together with bafilomycin (20 nM) for additional 24 hr before harvest. DMSO was used as a control. Cells were lysed with Triton lysis buffer (20 mM Tris-Cl, pH 7.5, 1 % Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM β-glycerophosphate, 2,5 mM sodium pyrophosphate, 1 mM Na- orthovanadate) supplemented with complete protease inhibitor cocktail (Roche, Basel, Switzerland), followed by centrifugation and samples were subjected to SDS-PAGE analysis and immunoblot for pan-phosphotyrosine (pTyr-ZNRF3), HA (total ZNRF3) and α-tubulin. For the quantitative analysis, background signals (cells without Dox treatment) were subtracted from phospho-tyrosine signals before normalizing to total ZNRF3. Dot graph was generated with Graphpad (Prism) using average pTyr signal and p-value (student t-test).