Anti qki

To extract the protein from patient samples, normal and tumor tissues were homogenized in RIPA buffer containing 50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA-free protease inhibitor cocktail (Roche, Germany) plus 1.5 mM phenylmethylsulfonyl fluoride (PMSF). Lysates were collected following the removal of insoluble material from tissue extracts by centrifugation at 14,000 rpm for 20 min at 4°C. Protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by gel transfer to a nitrocellulose membrane (BioRad, USA). The membranes were incubated first with the primary antibodies, and then with secondary antibodies coupled to horseradish peroxidase (HRP). Band signals were detected with an enhanced chemiluminescence (ECL) system (Thermo Scientific) and visualized by image analyzer (Fujifilm, Japan). The primary antibodies used for this study are anti-GAPDH (Kang Cheng Bio-tech, China), anti-QKI (Sigma, USA), anti-γ-tubulin (Sigma, USA), anti-FLAG (Sigma, USA), anti-NUMB (Cell Signaling, USA), anti-NICD (Val1744, Cell Signaling, USA), and anti-HA (Roche, Germany). The HRP-conjugated secondary antibodies anti-mouse IgG and anti-rabbit IgG were purchased from Promega.