Fig. 3.

Confirmation of LC-MS/MS results. A, B, equal concentrations of WCL and equal concentrations of EXO purified from DKs-8, DLD-1, and DKO-1 cells were resolved via SDS-PAGE and Western blotted with the indicated antibodies (for protein concentrations, see “Experimental Procedures”). C, trend analysis of proteins Western blotted in B. A trend analysis using the Jonckheere–Terpstra trend test was performed, and results for the indicated proteins were plotted. The y-axis indicates normalized counts, which are equivalent to the observed counts divided by the total number of confident identifications. The results follow the trend shown in B for ITGA6 (IPI00010697), ITGB4 (IPI00220845), EPHA2 (IPI00021267), and EPS8 (IPI00290337). D, KRAS is present in WCL and EXO. Proteins from DKs-8 and DKO-1 WCL and EXO (see supplemental Experimental Procedures) were resolved via SDS-PAGE and subjected to targeted LC-MRM analysis for WT (LVVVGAGGVGK) and mutant (LVVVGAGDVGK) (G13D) KRAS peptides. Concentrations of WT and mutant KRAS peptides were normalized to protein input and reported as fmol/μg protein. All experiments were performed at least in triplicate. E, equal protein concentrations of exosomes were resolved via SDS-PAGE and then gel stained with Sypro Ruby (see supplemental Experimental Procedures). Below, densitometry was performed on three biological replicates of DKs-8, DLD-1, and DKO-1 exosomes. Integrated densities of the entire lane were determined, and mean values (ID) and the S.D. were calculated. No statistically significant differences were found. The results show equivalent levels of exosomal proteins in the three preparations.