In vitro characterization of MEC-1 cells overexpressing WT and mutant BTK. GFP-labeled MEC-1 cell lines stably overexpressing mutant BTK (BTK^C481S or BTK^C481R) and BTK^WT were generated by using standard lentiviral transduction methods. Cells were sorted to enrich the transduced GFP⁺ cell populations in each cell line. For all experiments, a population of cells >75% GFP⁺ was used. (A) Phosphorylated (Y223) and total BTK proteins were overexpressed in transduced cells. p-PLCγ2 (Tyr1217) and p-ERK (Thr202/Tyr204) levels were also increased in all BTK-overexpressing cells. Vinculin was the loading control. (B) IgM stimulation in transduced MEC-1 cells. Cells (5 ×10⁵ per milliliter) were seeded in flasks, with or without IgM (20 µg/mL) and incubated at 37°C for 15 minutes. Protein extracts were subjected to immunoblot assays to determine the levels of pBTK (Y223), BTK, pERK (Thr202/Tyr204), and ERK. Vinculin was the loading control. (C) Exponentially growing GFP⁺ cells were seeded in flasks and incubated with ibrutinib (0.01, 0.1, and 1 µM) for 3 hours. Protein extracts were subjected to immunoblot assays to determine the levels of pBTK (Y223), BTK, pERK (Thr202/Tyr204), and ERK. Vinculin was the loading control. (D) Validation of cell surface markers. Cells were stained with anti-CD19-PECy7 and anti-CD23-PE monoclonal antibodies and analyzed with a flow cytometer. Flow cytometry dot plots showing GFP⁺ cells (left) and CD19⁺CD23⁺ cells (right) after gating on the GFP⁺ cell population.