Fig. 2
Mac-1’s αI domain interacts in cis with FcγRIIA’s ectodomain to lower FcγRIIA’s affinity for IgG. a Model of FcγRIIA, IgG, and Mac-1 in bent/closed and extended/open conformation based on published crystal structures. b FLIM was conducted on Jurkat cells expressing Mac-1 and FcγRIIA by staining with the donor (D) antibody for anti-FcγRIIA (IV.3 AF488) and the acceptor (A) antibody for Mac-1 (ICRF44 AF568) or an isotype control. FLIM images were taken and presented in pseudocolors from red to green. The fluorescence lifetimes of the donor in the absence (D) or presence (A + D) of the acceptor antibody were calculated. n = 3 experiments. *p < 0.01; unpaired t-test. c–f BFP measurements of adhesion frequency-versus-contact time of the indicated Jurkat cells (J-IIA) alone or with wild-type Mac-1 (WT), or Mac-1 lacking E253-R261 (E MT) with or without NIF treatment against bead immobilized Mac-1. c bead immobilized FcγRIIA (d) or bead immobilized IgG (e, f). The absence or presence of binding after a contact of given duration was analyzed to obtain the adhesion frequency. Bar graphs show effective 2D affinity (AcKa, E) and off-rate (koff, F) for each cell line. The adhesion frequencies of J-IIA-WT were lower than those of J-IIA, J-IIA-E MT, and NIF treatment of J-IIA-WT increased the effective FcγRIIA 2D affinity. Data is average ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, by unpaired, two-tailed Student’s t-test
