Everyblot blocking buffer

Whole-cell protein was extracted from flash-frozen PDX tumor tissue. Tissue samples were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors (Roche, cat no. 04693159001, 04906837001) using TissueLyser II (Qiagen) homogenizer at 4°C following the manufacturer's instructions. Samples were then centrifuged for 10 minutes at 15,000 × g and supernatant was collected for the assay. Protein concentration was measured by the Pierce BCA protein assay (Thermo Fisher, cat no.23225). Samples were separated on 4% to 20% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad) after heat denaturation in Laemmli sample buffer, and proteins were transferred to nitrocellulose membranes. Membranes were blocked with EveryBlot Blocking Buffer (Bio-Rad, cat no. 12010020) for 10 minutes at room temperature. The membrane was probed overnight at 4°C with the following antibodies: Myc (1:1,000; Cell Signaling Technology, 5605S), cyclophillin A (1:1,000, Cell Signaling Technology, 2175s) followed by incubation with secondary antibody [Li-Cor, IRDye 800CW Goat anti-Rabbit IgG (1:15,000) and IRDye 680RD Goat anti-Mouse IgG (1:15,000)] in EveryBlot Blocking Buffer for 1 hour at room temperature. Following washes in TBST, membranes were imaged using the Bio-Rad ChemiDoc MP imaging system.