Fd006

The radio immunoprecipitation assay lysis buffer (RIPA) (P0013B, Beyotime, China) containing a protease-inhibitor cocktail (HY-K0010, MCE, USA) were used to extract proteins from cells. The proteins were lysed in SDS-loading buffer (FD006, Fdbio, China), then 20μg proteins were resolved on a 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membrane (IPVH00010, Millipore, USA). The membrane was incubated with primary antibodies against PD-L1(13684, Cell Signaling Technology, USA), FLAG (80010-1-RR, Proteintech, USA), STT3A (12034-1-AP, Proteintech, USA), c-Jun (AF6089, Affinity, USA), HA (51064-2-AP, Proteintech, USA) and GAPDH (60004-1-Ig, Proteintech, USA) at a dilution of 1:1000, followed by incubation with species-specific (rabbit or mouse) HRP-conjugated secondary antibodies at a dilution of 1:5000. The immunoreactive bands were visualized by Omni ECL reagent (SQ101, EpiZyme, China).

The proteins were extracted from the samples using the radio immunoprecipitation assay lysis buffer (P0013B, Beyotime, China), containing a protease-inhibitor cocktail (HY-K0010, MCE, USA). The proteins were lysed in SDS-loading buffer (FD006, Fdbio, China), then the lysates were resolved on sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane (IPVH00010, Millipore, USA). The membrane was incubated with polyclonal antibodies against EGFR (AF6043, Affinity, China), phospho-EGFR (AF3047, Affinity, China), MMP2 (AF0577, Affinity, China), MMP9 (AF5228-MMP-9+Antibody.html">AF5228, Affinity, China), VE-Cadherin (AF6265, Affinity, China), AKT (bsm-33282M, Bioss, China), phospho-AKT (bs-0876R, Bioss, China), Foxq1 (PA1-31951, Invitrogen, USA) or GAPDH (AC033, Abclonal, China) at a dilution of 1:1000, then incubated with species-specific HRP-conjugated secondary antibodies at a dilution of 1:5000. The immunoreactive bands were visualized by enhanced chemiluminescence (WBKLS0100, Millipore, USA). phosphatase inhibitor cocktail (HY-K0021, MCE, USA) was used for the phosphoprotein blots, e.g., p-EGFR and p-AKT.

Cells were lysed with lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% TritonX-100) supplemented with protease inhibitor cocktail (TOPSCIENCE, Shanghai, China, #C0001) and phosphatase inhibitor cocktail I (TOPSCIENCE, Shanghai, China, #C0002) for 30 min. After centrifugation at 14,000 g and 4°C for 10 min, supernatants were collected and boiled for 5 min together with 5 × loading buffer (FDbio, FD006) to be used to perform SDS-PAGE. Each sample was loaded with 20–40 μg protein. Proteins were further transferred onto 0.22 μm PVDF membrane (Roche, 3010040001). The membrane was blocked with 5% fat-free milk in Tris-buffered saline added 0.1% Tween-20 (TBS-T) for 1–2 h at room temperature and then incubated with appropriate primary antibody at 4°C overnight. The membrane was washed three times for 10 min each with TBS-T and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. After washing three times with TBS-T, the membrane was flushed with FDbio-Pico ECL (FDbio). ChemiDoc Touch (Bio-Rad) achieved visualization. The uncropped scans of blots are shown in Supplementary Figure 3.