Inpp5k inactivation alters IL-7R and IL-7R–bound JAK kinases dynamic structure. (A) An indirect FRET strategy was used to analyze the proximity between IL-7Rα and γc ectodomains (left) and between JAK1 and JAK3 (right). Primary antibodies directed against IL-7Rα and γc ectodomains or against JAK1 and JAK3 as well as fluorophore-labeled secondary antibodies that specifically recognized the constant part of one of the primary antibodies were used. The green marker attached to the first secondary antibody represents the donor fluorophore (AF488), and the red one attached to the other secondary antibody represents the acceptor fluorophore (AF546). The closer the distance between the 2 probed proteins (IL-7Rα and γc ectodomains; JAK1 and JAK3), the higher the energy transfer and the resulting FRET signal (FRET efficiency). (B-C) Indirect FRET analysis of IL-7Rα/γc ectodomains (B) and JAK1/JAK3 (C) proximity in sorted B220⁺CD43⁺ bone marrow cells from control (WT, red columns) and VAV-CRE (blue columns) mice, before and after addition of IL-7 (2 ng/mL) for 2 minutes at 37°C. (B) Representative confocal pictures (original magnification ×63) of energy transfer (FRET efficiency) between AF488 (γc) and AF546 (IL-7Rα) labeled secondary antibodies. FRET efficiency was calculated in accordance with the indirect FRET method validated by Guala et al (left).²³ The rainbow scale represents the energy transfer between the 2 fluorophores, from 0.006 (blue) to 0.012 (white). The quantitative energy transfer between the 2 indirectly labeled γc and IL-7Rα ectodomains (FRET efficiency) is presented (right). Results represent mean ± SEM (n = 148-220 cells analyzed per group, from 6 mice). (C) Representative confocal pictures (original magnification ×63) of energy transfer (FRET efficiency) between AF488 (JAK3)- and AF546 (JAK1)-labeled secondary antibodies (left). FRET efficiency was calculated as described earlier. The rainbow scale represents the energy transfer between the 2 fluorophores, from 1 (blue) to 3 (white). The quantitative energy transfer between indirectly labeled JAK3 and JAK1 (FRET efficiency) is presented (right). Results represent mean ± SEM (n = 180-242 cells analyzed per group, from 6 mice). P values were calculated using unpaired nonparametric t test. NS: P > .5; ∗P < .5; ∗∗P < .1; ∗∗∗P < .01; ∗∗∗∗P < .001. NS, nonsignificant; SEM, standard error of the mean; WT, wild type; FRET, fluorescence resonance energy transfer.