Extensive preclinical validation of combined RMC-4550 and LY3214996 supports clinical investigation for KRAS mutant pancreatic cancer. Frank et al. Uncrossed Western Blot
Uncrossed western blot pictures from the paper: "Extensive preclinical validation of combined RMC-4550 and LY3214996 supports clinical investigation for KRAS mutant pancreatic cancer" Frank et al., Cell Reports Medicine.
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To prepare analysis of cell lysates, cells were plated in complete medium. The morning after, cells were refreshed with medium and drugs of interest. At the desired time points, the cells were washed with cold-PBS and lysed in RIPA buffer supplemented with HaltTM Protease & Phosphatase single-use inhibitors cocktail (100 X) (78442) and HaltTM Protease single-use inhibitors cocktail (100 X) (78430). For preparation of tissue lysates, PDAC tissue from mice was homogenized in MLB Buffer containing protease inhibitor (Serva) and phosphatase inhibitor cocktails (Serva). Protein quantification was performed with the BCA Protein Assay Kit (Pierce). The lysates were then resolved by electrophoresis in Bolt 4 – 12 % Bis-Tris Plus Gels (Thermo Fisher Scientific) followed by western blotting as described previously (23). The following antibodies were used: Antibodies against RSK-1 (8408), phosphorylated RSK-1 (9344 and 8753) and GAPDH (5174) were purchased from Cell Signaling Technology (CST). Antibodies against alpha Tubulin (T9026) and Vinculin (V9131) were purchased from Sigma-Aldrich.