Figure 5: ABCA1 interactions with BTN3A1, apoA-I and IPP.
(a) ABCA1 and BTN3A1 co-immunoprecipitate in untreated and ZA-treated DCU937 and DC but not in CD14+ cells, and ZA treatment does not modify BTN3A1 expression. Pancadherin is employed as a control of equal protein loading (one out of three blots). (b) PLA of ABCA1-BTN3A1 interaction by confocal laser-scanning microscopy (ocular lens: × 10; objective: × 63). Ctrl: cells without primary antibodies. Scale bar, 10 μm; blue: nuclear staining (DAPI); green: ABCA1/BTN3A1 interaction (one out of the three experiments). (c) apoA-I is physically associated with ABCA1, not with BTN3A1. The expected molecular weight of apoA-I, ABCA1/apoA-I and BTN3A1/apoA-I are shown (one out of the three experiments). (d) ABCA1 and BTN3A1 expression in DC after incubation with siRNA for Abca1 (siABca1), Btn3a1 (siBtn3a1) or with scrambled non-targeting siRNA (scr). β-tubulin was employed as control of equal protein loading (n=3). (e) IPP is physically associated with ABCA1 in untreated and ZA-treated DC. The two bands of 150 and 100 kDa in the dithiothreitol (DTT)- and trypsin-treated DC lane are detected with an antibody specific for the N-terminal extracellular loop of ABCA1 in immunoblotting (IB; left). Autoradiography signal of the IPP-ABCA1 interaction (right). Pancadherin is employed as a control of equal protein loading (one out of three experiments). (f) Extracellular IPP release in Abca1- and/or Btn3a1-silenced DC left untreated (ZA-) or after ZA treatment (ZA+). The bars represent the mean±s.e.m. of three experiments (**P<0.001, Wilcoxon–Mann–Whitney). (g) Vγ9Vδ2 T cell proliferation after 7-day PBMC stimulation with supernatants from Abca1- and/or Btn3a1-silenced DC. The bars represent the mean±s.e.m. of three experiments (**P<0.01; Wilcoxon–Mann–Whitney).
