Anti cd274

Total proteins of thecells were extracted by lysis buffer (Tris-HCl, PH 8.0, 400 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1 mM Na pyrophosphate, 1% Triton X-100, 10% glycerol), with supplement of protease inhibitors (Roche, Indianapolis, IN). The protein concentration was determined by a BCA kit (Piece, Rockford, IL). Then protein was loaded onto 12% SDS-PAGE gel and was electronically transferred to PVDF membranes (Millipore, Billerica, MA). Primary and secondary antibodies were then incubated to the membranes. The HRP-conjugated secondary antibody (1:5000, Sigma-Aldrich) was incubated for 2 h atroom temperature. Protein signals were detected via ECL method. The primary antibodies used in this study includes anti-BRD4 (1:1000, Abcam), anti-c-Myc (1:1000, Abcam), anti-CyclinD1 (1:1000, Abcam), anti-CDK4 (1:1000, Abcam), anti-MMP2 (1:1000, Abcam), anti-MMP9 (1:1000, Abcam), anti-CtIP (1:1000, Abcam), anti-CD274 (1:1000, Abcam), anti-KRAS (1:500, Abcam), anti-E-cadherin (1:10000, Abcam), anti-N-cadherin (1:1000, Abcam), anti-Vimentin (1:3000, Abcam) and anti-GAPDH (1:1000, Abcam). All the primary antibodies were incubated for 4 h at 37 °C.

Western blotting was performed rely on relevant protocol. Proteins were transferred onto PVDF membranes (ThermoFisher, USA), and incubated 8 h in the 4°C fridge with the following primary antibodies: Anti‐IKBKE (Cell Signaling, USA, 1:1000), Anti‐CD274 (Abcam, USA, 1:1000), Anti‐pSTAT3 (Tyr705) (Cell Signaling, USA, 1:1000), Anti‐STAT3 (ABclonal, UK, 1:1000), Anti‐GAPDH (ZSGB‐Bio, China, 1:2000). Secondary antibodies: HRP labeled goat anti‐rabbit/mouse IgG (ZSGB‐Bio, China, 1:1000). Chemiluminescent HRP substrate (Millipore, USA) and GBOX system (Syngene Company, UK) were used to detect protein expression.