Figure 6.

Galectin-9 interacts with the SNX3 C-terminus and regulates borreliae compaction. (A) Western blots of anti-GFP immunoprecipitations of lysates from macrophages expressing GFP as control or GFP-SNX3-C construct, developed with GFP-, galectin-9–, or B. burgdorferi–specific antibodies, with load (ld) and elution (el) fractions, as indicated. (Note that galectin-9 blot was reprobed for detection of borreliae. Comparison between respective lanes of SNX3-C elutions thus shows that the galectin-9 signal is not due to overspill of the load lane.) Molecular weight is indicated in kilodaltons. (B–F) Endogenous forms of SNX3 and galectin-9 localize to borreliae phagosomes. Confocal micrographs of macrophage stained for SNX3 and galectin-9, borreliae stained with B. burgdorferi–specific antibody. Yellow boxes indicate detail regions shown enlarged below, with merges (C1–F1, C2–F2, and C3–F3). (G) GFP-galectin-9 vesicles contact borreliae phagosomes. Shown are still images from live-cell video of macrophage expressing GFP-galectin-9 and RFP-SNX3, with borreliae and nucleus stained by Hoechst 33342 (Video 4). Yellow box indicates detail region shown enlarged in galleries (G1–G4) below. Time since start of experiment is indicated in minutes:seconds. Dotted circles indicate contact of galectin-9 vesicles with borreliae phagosomes. Scale bars: 10 µm for B and G, and 2 µm for insets. (H) Western blot of lysates from macrophages treated with control siRNA, SNX3-specific or galectin-9–specific siRNA, or a combination of both. Blots were developed with SNX3- or galectin-9–specific antibodies or with pan-actin antibody for loading controls. Molecular weight in kilodaltons is indicated on the left. (I) Evaluation of borreliae compaction in cells treated with control siRNA, SNX3 siRNA, or galectin-9 siRNA or a combination thereof. Values are given as mean ± SEM. Note the absence of additive effect in SNX3 and galectin-9 double knockdown. For specific values, see Table S1. n = 3. One-way ANOVA; *, P < 0.05.