V5 epitope

Fresh resected human LUAD and matched adjacent noncancerous tissue were obtained after informed consent and approval by the institutional review board (16-1514, 16-107, and 12-245) at Memorial Sloan Kettering Cancer Center. Human LUAD cell lines, human lung fibroblast cells and a normal bronchial epithelial cell line (NL20) were obtained from American Type Culture Collection; Lenti-X 293T cells were purchased from Takara Bio. All cells were tested for mycoplasma. The primary antibodies used were BRMS1 (Abcam, ab134968), c-fos (Cell Signaling, #2250), c-Jun (Cell Signaling, #9165), V5-epitope (Abcam, ab15828), CEACAM6 (Santa Cruz, SC-59899), Src (Cell Signaling, #2109), p-Src Y416 (Cell Signaling, #59548), p-fos S32 (Cell Signaling, #5248), actin (Santa Cruz, sc-8432), p-Jun S63 (Cell Signaling, #2361), RAMP1 (Thermo Fisher, 10327-1-AP), and F5 (Thermo Fisher, PA5-103046). The reagents used—c-fos inhibitor T5224 (S8966) and Src inhibitor Saracatinib (AZD0530, S1006)—were purchased from Selleck Chemicals. Collagen Type IV (C6745), Collagen Type I solution (C3867), and TCN (T7760) were purchased from Millipore Sigma; puromycin (A1113803) and blasticidin (A1113903) were purchased from Thermo Fisher; CellTiter-Glo was obtained from Promega (G9241); and poly-HEMA solution was purchased from Sigma-Aldrich (P3932).

A375 cells were exposed to DOX with or without VX-11e (2 μmol/L), or trametinib (3 nmol/L), then harvested in RIPA buffer, and run on SDS-PAGE. Proteins were analyzed using the following antibodies: phosphoERK1^T202/Y204/ERK2^T185/Y187, ERK1/2, phosphoELK-1^S383 (Santa Cruz Biotechnology), phosphoRSK^S380, RSK, tubulin, DUSP6 (Cell Signaling Technology), cyclin D1 (Thermo Scientific), and V5 epitope (Abcam). Relative expression was calculated by dividing the relative densities of each phoshoELK-1 band by total ERK (or V5) using Image J. Values were normalized to untreated wild-type, which was set at 1.