Expression and activation of Lyn and Btk in neoplastic MCs. (A) Immunocytochemical detection of p-Lyn^Tyr507, p-Lyn^Tyr396, and p-Btk^Tyr551 as well as total Lyn and total Btk in HMC-1.1 (top panels) cells and HMC-1.2 (bottom panels) cells. (B) Western blot analysis of Btk and Lyn expression in various cell lines using Abs against p-Lyn^Tyr507, Lyn, p-Btk^Tyr223, Btk, and β-actin as indicated. Molecular size markers are also shown. (C) Immunohistochemical detection of tryptase, p-Lyn^Tyr507, Lyn, p-Btk^Tyr551, and Btk in patients with indolent systemic mastocytosis (ISM), aggressive SM (ASM), and mast cell leukemia (MCL). The method of staining is provided in “IHC and ICC.” Figures shown in panels A and C were prepared using an Olympus DP11 camera connected to an Olympus BX50F4 microscope equipped with 100×/1.35 UPlan-Apo objective lens. Images were prepared using Adobe Photoshop CS2 Version 9.0 software (Adobe Systems) and processed with PowerPoint software (Microsoft). (D-E) Detection of p-Lyn^Tyr507, p-Src^Tyr416, and p-Btk^Tyr223 in primary neoplastic cells by Western blotting. Neoplastic cells were obtained from patients with ISM (n = 3) and ASM (n = 3; D) as well as from patients with acute myeloid leukemia (AML, n = 5), chronic myeloid leukemia (CML, n = 1), and chronic neutrophilic leukemia (n = 1; E). In addition, normal BM cells (NBM, control) and HMC-1.2 cells (positive control) were examined (E). The β-actin control as well as size markers are also shown.