Mutants NTRK2^A203T, NTRK3^E176D, and NTRK3^L449F increase Trk cell-surface localization but not retention. (A-B) To assess differences in mutant receptor localization, WT and mutant Ba/F3 cells were stained with APC-conjugated anti-TrkB or anti-TrkC antibodies and analyzed by flow cytometry. A significantly higher localization of Trk receptors was observed with NTRK2^A203T, NTRK3^E176D, and NTRK3^L449F mutants relative to their respective WT receptor. Validation of APC-conjugated Trk flow antibodies is shown in supplemental Figure 9C-D. (C-D) To assess receptor retention at the cell surface, WT and mutant NTRK2 and NTRK3 Ba/F3 cells were treated with 100 µg/mL cycloheximide for 12 or 24 hours, stained with APC-conjugated anti-TrkB or anti-TrkC antibodies, and analyzed via flow cytometry. Following 12 hours, NTRK2^A203T was less stable than NTRK2^WT. Both NTRK3 mutants were less stable than NTRK3^WT after 12 and 24 hours of cycloheximide treatment. Normalized mean fluorescence intensity (MFI) is shown over time. For all experiments, WT Ba/F3 cells were grown in IL-3–supplemented media and all lines were starved overnight in 0.1% BSA RPMI. Staining was performed in triplicate. Statistical significance was assessed by a 1-way or 2-way ANOVA followed by a Tukey multiple comparison test. All statistical comparisons shown are between WT and mutant receptors. The average mean plus or minus SEM is shown. *P < .05; **P < .01; ***P < .001; ****P < .0001.