S100A8 locates at the IST1-structures. (A) Confocal micrographs of moDCs immunolabeled for IST1 or CHMP1B (magenta) and a set of other proteins identified by mass spectrometry (green). The choice for which proteins were further investigated was based on the availability of antibodies and the function of the identified protein. Calprotectin subunit s100a8 localized in the IST1 structures, while the other subunit, s100a9, did not. The MHC class I form HLA-C did not co-localize with IST1 structures, except for one observation in one donor (bottom zoom), pointing at absent or very transient co-localization with IST1 structures. Neither the RNA stain SYTO RNASelect nor the DNA/RNA binding proteins Histone H3 and HNRNPC, nor the nuclear protein calcineurin-binding protein cabin-1 co-localized with IST1 structures. We also could not confirm the presence of cytoskeleton-involved proteins like actin related protein 2/3 complex subunit 2 (ARPC2), which is the actin-binding component of the Arp2/3 complex mediating actin polymerization, nor myosin-9, which plays a role in focal contact formation (Betapudi, 2010). Synaptogyrin-2, which may play a role in exocytosis in rat cells (Sugita et al., 1999), or tapasin, which is involved in the association of MHC class I with peptide and transporter associated with antigen processing (TAP), could also not be found in the structures. Proteins identified by the IST1-pulldown but not present in IST1-positive structures might bind IST1 in other subcellular locations. Scale bars: 10 μm.