Ets 1

BRAF mutant A375 parental and vemurafenib-resistant cells were grown on 6-well slides for 24 h. Media was then decanted and the wells were washed 3 × with PBS. Cells were fixed in methanol for 20 min at −20 °C, washed 3 × with PBS and blocked for 1 h at room temperature in PBS with 0.3% Tween-20 and 5% BSA. The primary antibody was Ets-1 (Bethyl), which was diluted 1:100 in PBS with 0.3% Tween-20 and 1% BSA (antibody dilution buffer) and incubated overnight at 4 °C. After 3 × washes in PBS, Alexa Fluor 488 anti-Rabbit secondary antibody (Life Technologies) was added at 1:500 in antibody dilution buffer and with DAPI incubated for 1 h at room temperature. After 5 × washes in PBS, slides were coverslipped with ProLong Gold anti-fade reagent (Invitrogen). Images were acquired using an Olympus Fluo View 500. A representative image from each sample is shown.

Cells were lysed in RIPA and the proteins were subjected to an SDS-PAGE and western blot analysis was carried out according to standard protocols using the following antibodies: TERT (Santa Cruz technology and Thermo Fisher Scientific), PhosphoERK (Sigma), ERK (Millipore), PhosphoETS1 (Sigma), ETS1 (Bethyl) and GABPA (Santa Cruz technology). Antibodies were visualized using the SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher Scientific).

Primary antibodies used in this study include: NRAS, Pan-RAS, HRAS, Dusp4, Ubiquitin (total), Ets-2, GABPAα, Mcl-1, HA, β-Actin, ERK2 (total) (Santa Cruz); KRAS (Calbiochem, OP24); AF-6, Usp9x, Ets-1 (Bethyl Laboratories); pERK, Caspase8, PARP, BID, BIM (Cell Signaling); ERG (Abcam); HA (Roche); FLAG (Sigma). Blots were developed with ECL substrate (Pierce) and imaged on X-ray film (BioExpress). Antibody catalogue numbers and their dilutions are included in Supplementary Table 2.