Aliquots of frozen, powderized patient-derived xenograft tumors from established basal (WHIM2, passage 32) and luminal A (WHIM16, passage 33) breast cancer subtypes were obtained from the Washington University Clinical Proteomic Tumor Analysis Consortium (CPTAC) Proteome Characterization Center. The xenografts were raised subcutaneously in 8 week old NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice (Jackson Labs, Bar Harbor, Maine) as previously described [29 (link), 45 (link)]. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Washington University in St. Louis, MO.
Two aliquots of xenograft tumor tissue from basal subtype and two aliquots of xenograft tumor tissue from luminal subtype were used. Tissues were sonicated in denaturing buffer (8 M urea, 50 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA, 50 μM PR-619, 1 mM 2-chloroacetamide) supplemented with protease inhibitors (2 μg/mL aprotinin, 10 μg/mL leupeptin, 1 mM PMSF). One hundred μL denaturing buffer was added per 10 mg tissue. Lysates were sonicated and cleared by centrifugation at 16,000 x g for 20 min at 4 °C.
Two aliquots of xenograft tumor tissue from basal subtype and two aliquots of xenograft tumor tissue from luminal subtype were used. Tissues were sonicated in denaturing buffer (8 M urea, 50 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA, 50 μM PR-619, 1 mM 2-chloroacetamide) supplemented with protease inhibitors (2 μg/mL aprotinin, 10 μg/mL leupeptin, 1 mM PMSF). One hundred μL denaturing buffer was added per 10 mg tissue. Lysates were sonicated and cleared by centrifugation at 16,000 x g for 20 min at 4 °C.
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