Page gels

Mutational analysis of exon 5 of GNAQ and GNA11 was conducted in a CLIA certified laboratory. Standard PCR amplification of a 250bp and 245bp fragment for GNAQ and GNA11, respectively, including the entire coding region of exon 5, was performed in duplicate using HotStar Taq DNA polymerase (Qiagen) and primers listed in eTable 2. PCR was also performed using standard primers with a 10–mer locked nucleic acid (LNA) oligonucleotide, designed to suppress amplification of wild-type DNA. Sequencing and analysis were performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) on an ABI3730 running ABI Prism DNA Sequence Analysis Software.
Western blotting was performed for pERK and cyclinD1, and quantitated by densitometry using ImageJ software. Cells were lysed in radioimmunoprecipitation assay buffer supplemented with protease inhibitor cocktail tablets (Roche Diagnostics) and 1 mmol/L Na3VO4. Equal amounts of protein were loaded on 4% to 12% PAGE gels (Invitrogen). Polyvinylidene difluoride membranes were blocked with 5% nonfat dried milk and probed with pERK, ERK, cyclin D1, and α-tubulin (Cell Signaling). Wilcoxon rank sum test was used to evaluate associations between radiographic regression (RECIST response or stable disease of >16 weeks) and suppression of pERK and cyclin D1.