BiLC fusions are expressed.

(A) Map detailing lentiviral expression constructs for BiLC fusions, stably expressed in U87MG cells. Cartoon depicts how these fusions were stably expressed in cells for co-expression (CE) and co-culture (CC) experiments. LTR = long terminal repeat, WPRE = woodchuck hepatitis virus post-transcriptional regulatory element, T2A = self-cleaving peptide, PuroR = puromycin resistance cassette, BSD = blasticidin resistance cassette, Tet-On 3G = transactivator protein, EF1a = translation elongation factor 1a promoter, TRE3GV = Tet-On 3G system promoter. (B, C) Lysates from cell-normalized equivalents from co-cultured (CC) or co-expressed (CE) BiLC fusions stably expressed in U87MG and grown in media +/- Dox (250 ng/ml) (using conditions detailed for the BiLC experiment in Materials and Methods) were resolved on an SDS gel and immunoblotted for (B) nLuc-HA-K-Ras (anti-HA) or (C) cLuc-Flag-Raf-RBD (anti-Flag) with tubulin levels used for normalization (black arrows indicate protein of interest). K-Ras mutations analyzed are indicated above lane(s). Irrelevant lanes on the same gel are covered with a box. Complete, uncropped blots are available in S3 Fig (D) BiLC K-Ras fusion protein expression in lysates from 1.0 x 105 cells (cell lysate), and from lysates of pellets of conditioned media: produced from 3.7 x 107 cells (16,500xg pellet = P16.5Kxg) and from 2.1 x 107 cells (110,000xg pellet after removing 16,500xg pellet and 0.2 μm filtration = P110Kxg). Color-coded arrows show protein bands of interest from a scan of an immunoblot probed with the indicated antibodies on the Li-Cor Odyssey using secondary detection antibodies labeled with two different colors. Integrin α5 was used as a normalization control. Bracket shows truncated fragments of full-length fusion protein. Separate channel images in grayscale are available in S3 Fig (E) Total background-subtracted signals for the indicated K-Ras fusion protein’s expression in exosomes purified from U87MG cells stably expressing the indicated constructs were normalized to integrin α5 signal. Whole cell lysate signals were calculated in the same manner, then multiplied by a factor to adjust for the diluted number of cells represented by this signal as compared to exosomes. Normalized exosome signals/whole cell lysate signals x 100 produced the percentages for each data point. Individual experimental data points are indicated as symbols. For nLuc-HA-K-Ras analysis: (D) and an additional independent experiment were used (a total of 2 independent experiments); calculations available in S2 Table. For Flag-K-Ras analysis: 3 independent experiments were used from a prior publication characterizing Ras release to exosomes [1]; calculations available in S3 Table.