The highly motile feature of FcγRIIB is linked to its potent inhibitory function. (A) Shown are the schematic illustrations for the amino acid sequence of the TM domains of FcγRIIB-I232, FcγRIIB-T232, and FcγRIIB-CD86-TM. (B and C) Single-molecule imaging experiments were performed on A20II1.6 B cells expressing FcγRIIB-I232, FcγRIIB-T232, or FcγRIIB-CD86-TM to compare the Brownian motility of these FcγRIIB variants. Given are CPD plots (B), MSD plots (C, left), and scatter plots (C, right), respectively. Bars represent median value. The p-value between FcγRIIB-T232 and FcγRIIB-I232 (or FcγRIIB-CD86-TM) is <0.0001 in Kolmogorov-Smirnov tests. The results shown are representative of one of at least three independent experiments. (D and E) Ratiometric calcium flux in A20II1.6 cells expressing FcγRIIB-I232, FcγRIIB-T232, or FcγRIIB-CD86-TM that were stimulated with 5 µg/ml whole IgG anti–mouse Igκ (D) or F(ab’)₂ fragment anti–mouse Igκ (E). The results shown are representative of one of three independent experiments.