Anti kras

The KRAS^G12D expression of organoids was determined by western blotting analysis. After dissociating organoids from BME gel by TrypLE express and harvesting the cells, cells were washed with PBS buffer, then lysed in 0.5% Triton buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 5% glycerol, 0.5% Triton X-100, Protease Inhibitor (Sigma, P8340)), and centrifuged for 10 min at 4 °C. Total protein was quantified with BCA assay (Thermo Fisher, 23227). Equal amounts of the protein were mixed with 2× Laemmli sample buffer (BioRad, 161-0737), incubated at 95°C for 5 min, and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS‒PAGE). Western blotting was carried out following a standard protocol. The membrane was incubated with primary antibody in TBST buffer at 4°C overnight, followed by incubation with corresponding horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature, and then visualized using the SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher, 34580). The following antibodies were used: anti-KRAS at 1:1000 (Proteintech, 12063-1-AP); anti-RAS^G12D at 1:1000 (Cell Signaling Technology, 14429); anti-β-actin at 1:5000 (Sigma, A5441); goat-anti-rabbit IgG at 1:5000 (Jackson ImmunoResearch, 111-035-003), and goat-anti-mouse IgG at 1:5000 (Jackson ImmunoResearch, 115-035-003).

Proteins were isolated, subjected to SDS-page, transferred onto PVDF membranes and incubated with antibodies anti-SIAH1 (Abcam, Cambridge, MA, USA), anti-SFRP2 (Cell Signaling Technology, Danvers, MA, USA), anti-KRAS (Proteintech, USA), anti-Ki-67 (Abcam, Cambridge, MA, USA), anti-p27, anti-p21, anti-cyclinD1 (Bioworld Technology Inc. St. Louis Park, MN, USA). LamB1 and a-tubulin (Sigma, Saint Louis, MO, USA) were used as loading controls. Then, the membranes were detected by chemiluminescence. All operations above were performed in accordance with standard methods.