N ras

Tissue and cells were collected after washing with cold PBS and then lysed in RIPA lysis buffer (Solarbio) with protease inhibitor cocktail (Roche). Protein concentration was determined with a Lowry protein concentration detection kit (Solarbio). An equal amount of proteins for each sample was subjected to 8–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to nitrocellulose membranes. Antibodies were GATA3 (1:500, Proteintech), COTL1 (1: 200, Proteintech), N-Ras (1:500, Proteintech) and GAPDH (1:1000, Proteintech).

RMS cell lines were plated at a density of 1 × 10⁵ cells on glass coverslips in 6-well plates. Cells were incubated in 1000 nM tipifarnib for 24 h prior to washing in PBS and fixing in 4% paraformaldehyde at room temperature for 10 min. Cells were then incubated in blocking buffer (10% goat serum) for 1 hour at room temperature. Cells were incubated overnight at 4^oC in 1:100 dilutions of each HRAS (Proteintech, Catalog number: 18295-1-AP) and NRAS (Proteintech, Catalog number: 10724-1-AP) antibodies in blocking buffer (1% goat serum). Cells were then incubated in 1:1000 goat anti-mouse or goat anti-rabbit AlexaFluor488 with filamentous actin stain in blocking buffer for 1 h at room temperature in the dark. After staining, slides were counterstained with a 1:10,000 dilution of 50 µg/ml 40,6-diamidino-2-phenylindole (DAPI; Sigma Aldrich; # D9542; 5 mg/mL) for 5 min to visualize the nuclei of all cells. Coverslips were mounted with Prolong Diamond mounting medium reagent. After washing, cells were imaged on a Leica SP8 scanning confocal microscope with a ×63 oil immersion lens. All experiments shown were replicated at least twice.