Soft-agar colony formation assay and xenograft propagation. Soft-agar colony formation assay and xenograft propagation were carried out as described [22] and [23]. All animal procedures were performed according to Sulfo-NHS-SS-Biotin protocol approved by the institutional Animal Care and Use Committee at Hokkaido University Graduate School of Medicine.
Histological analysis and immunohistochemistry. Formalin-fixed paraffin-embedded tissues, including human glioma specimens and the KMG4-derived xenografts, were sectioned and stained with haematoxylin and eosin (H&E) using standard protocol. Immunohistochemistry was performed using anti-Crk (Transduction Laboratories, Lexington, KY, USA) and anti-Ki67 (MIB1; Dako, Glostrup, Denmark) antibodies.
Immunoprecipitation and immunoblotting. Protein determination, immunoprecipitation, SDS–PAGE and immunoblotting were carried out as described previously [9]. Antibodies were obtained from the following sources: anti-phosphotyrosine (PY20 and RC20H), anti-p130Cas, anti-paxillin, and anti-Crk antibodies (Transduction Laboratories, Lexington, KY, USA); anti-C3G (C19), anti-DOCK180 (H4), and anti-Crk-L (C20) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-actin antibody (Chemicon International, Temecula, CA, USA); anti-Flag (M2) antibody (Sigma).
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To monitor the DOM decomposition processes in bottles seawater
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