Functional characterization of the MAP2K1 mutants. (A) HEK293 cells were transiently transfected with expression plasmids encoding the indicated MAP2K1 and BRAF wild-type and mutant cDNAs, and corresponding lysates from cells maintained in serum were subjected to immunoblotting with the indicated antibodies. (B) Identification of functional implication of MAP2K1 mutations by IFC. Single cell suspension of LCH lesions were stained with Brilliant Violet (BV) 421-conjugated CD3, phycoerythrin (PE)-CD207, Alexa Fluor (AF) 488-phospho-MEK1, and AF647-phospho-ERK1/2 antibodies and analyzed by the gating strategy described in “Methods.” The top panels depict the distinctive CD3⁺ and CD207⁺ population sequestration (left) and overall p-MEK1⁺p-ERK1/2⁺ CD207⁺ (middle) or CD3⁺ (right) cells obtained by gating on 10 000 events. The middle and bottom panels show the representative images of random single cell events of 10 (138 through 2061) or 6 (308 through 4600) in CD207⁺ and CD3⁺ cells, respectively, identified by gating events with intermediate bright field (BF) area and BF aspect ratios close to 1. The indicated populations were then analyzed for coexpression of p-ERK1/2 or p-MEK1 in the CD207⁺ or CD3⁺ cells in the indicated channels. (C) Median fluorescent intensity (MFI) of p-MEK1 or p-ERK1/2 in CD207⁺ cells determined through IFC in patients harboring BRAFV600E (patients LCH5, LCH7, and LCH13) and MAP2K1 c.302_307del (patient LCH24) mutations, and patients with no somatic mutations (LCH30, LCH32, LCH37, LCH38, and LCH40).