Molecular biology grade dmso

Wild-type zebrafish strain AB/Tuebingen TAB-14(AB/TuebingenTAB-14) (Catalog ID: ZL1438) were obtained from zebrafish international resource center and grown in an animal facility at the Department of Zoology, King Saud University, Riyadh, Kingdom of Saudi Arabia. The fish were maintained and bred following guidelines of the Institutional Animal Care and Use Committee (ICUAC) and zebrafish book. The fertilized embryos were obtained by natural pairwise breeding of adult fish. The fertilized embryos were sorted, dead embryos were removed, and synchronous stage embryos were used for screening.
A stock solution of 25 mM was made by dissolving the compounds in molecular biology grade DMSO (Sigma Aldrich, St. Louis, MI, USA). Zebrafish embryos were exposed to serial dilution (1, 5, 15, 45, 150, and 300 µM) of each compound. The embryos were remained exposed to the compounds for 3 days, and the embryos medium containing the compounds were changed every day. The response of the embryos toward mortality, and embryonic toxicity (teratogenicity) was monitored once after 12 h and then after every 24 h until the end of the experiment. The experiment was repeated at least three times (triplicate biological repeats) by using a new batch of embryos every time. LC₅₀ for zebrafish embryonic toxicity was calculated by using an updated Probit analysis by Finney method [28 ].

All commercial chemicals and solvents were purchased from Sigma Aldrich or Fisher Scientific and used without further purification. The identity and purity of each product was characterized by MS, HPLC, TLC, and NMR. Purity of target compounds has been determined to be >95% by LC/MS on a Waters Autopurification system with PDA, MicroMass ZQ and ELSD detector and a reversed phase column (Waters X-Bridge C18, 4.6 × 150 mm, 5 µm) eluted with water/acetonitrile gradients, containing 0.1% TFA. Stock solutions of all inhibitors were prepared in molecular biology grade DMSO (Sigma Aldrich) at 1000× concentrations. The epiHSP70 ligands and probes, the epiHSP90 inhibitor PU-H71, the PU-beads and the control baits were generated using published protocols^{27 (link)–31 (link),80 (link)} and described in Supplementary Notes 1. Thymidine, nocodazole, RO-3306, MG132, paclitaxel, VER155008 and MKT077 were purchased from Sigma-Aldrich. Proteins were generated and purified using published protocols described in Supplementary Note 2^{81 (link)}.

Small molecule compounds were either purchased from ChemBridge Corporation or were synthesized (HepIn-13). HepIn-13 was prepared by heating 1,8-diaminonaphthalene (1 mM) and 4-bromobenzaldehyde (1 mM) overnight in absolute ethanol. The resulting mixture was concentrated and the resulting dark brown/purple solid was recrystallized twice from 95% ethanol to yield a light purple crystalline solid (64% yield). The product was homogeneous by HPLC (>95% purity) and had ¹H NMR and mass spectra consistent with the proposed structure. The chromogenic peptide pyroGlu-Pro-Arg-pNA (s-2366) was purchased from DiaPharma. Rabbit anti-human Hepsin polyclonal antibody was purchased from Cayman Chemical (#100022). Anti-Flag antibody was purchased from Sigma (F1804). Anti-HA antibody was purchased from Roche (#1867423). Goat anti-rabbit secondary antibody was purchased from Jackson ImmunoResearch. Human recombinant Matriptase was purchased from R&D Systems. Molecular biology-grade DMSO was purchased from Sigma. Purified recombinant human pro-HGF was obtained from Dr. George F. Vande Woude (Van Andel Research Institute).