CTCL cell line Hut78 harbors Q61K mutation for NRAS. (A) RNA was isolated and reverse transcribed to cDNA for all 4 cell lines. Next, PCR was performed covering the open reading frame of KRAS, NRAS, HRAS, and BRAF. PCR products were purified and sent for sequencing. Results were evaluated by Genetic Analysis Technology Consortium sequence viewer. Arrow indicates cytosine-to-adenosine conversion. (B) Table of sequencing results for all 4 CTCL cell lines. A heterozygous Q61K mutation was detected for the Hut78 cell line. A homozygous silent H25H mutation was found in the SeAx cell line. (C) All 4 cell lines were kept under equal conditions, lysed, and and lysates subjected to Western blot. Phosphorylation of ERK was assayed by anti-phospho-specific ERK antibodies. Equal expression of ERK was verified by anti-ERK2 antibodies, and equal loading was verified by anti–tubulin antibodies. Quantification of phosphorylated ERK to ERK2 was done with ImageJ 1.43 Quantification software. (D) Same as panel C, but anti-phospho-specific MEK antibodies and anti-MEK antibodies were used to assess the phosphorylation status of MEK and MEK expression, respectively. Equal loading was verified by antitubulin antibodies.