Figure 2: LGALS3BP localizes to centrioles.

(a) LGALS3BP localizes to the proximal part of centrioles in GFP-centrin-U2OS cells in interphase. The respective labelling with a monoclonal mouse LGALS3BP antibody is shown in red, GFP-centrin in green and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI)-stained DNA in blue in the composite images. Scale bars, 5 μm. (b) Flag-LGALS3BP and Flag-GFP as control were overexpressed in U2OS cells. Flag-LGALS3BP localizes to the centrioles upon pre-fixation extraction and labelling with an anti-Flag antibody (red), an anti-centrin antibody (green) and DAPI-stained DNA (blue). Flag-GFP does not show centriolar labelling. (c) Schematic LGALS3BP localization to the centrioles. (d) TEM immunogold localization of LGALS3BP detected with the mouse LGALS3BP antibody and a 5-nm gold-coupled secondary antibody in U2OS cells. Pre-embedding labelling (top row and bottom right image) and LR-gold post-embedding labelling independently confirmed LGALS3BP localization to the proximal part of centrioles as identified by procentrioles (PC) or presence of the distal centriole subtending appendages (Ap) on the opposite end. The negative control (non-specific IgG antibody) shows no gold labelling. Scale bars, 200 nm. (e) LGALS3BP resides near the proximal part of the distal basal body in rat sperm and testis cryosections. The three panels show single-plane confocal images of earlier and late stage rat sperm labelled with an anti-acetylated α-tubulin antibody (i), anti-centrin antibody (ii) and γ-tubulin antibody (iii) (green), co-labelled with a rabbit anti-LGALS3BP antibody (red). DAPI-stained DNA is shown in blue. Basal body localization of LGALS3BP is detected in earlier stages with centrioles still present (filled arrowheads) and in mature stages after their degeneration (empty arrowheads) as judged by lacking respective markers. (f) Localization of LGALS3BP was confirmed in TEM using pre-embedding labelling with rabbit anti-LGALS3BP and 10 nm gold-coupled secondary antibodies followed by ultrathin sectioning. One to four gold particles were consistently detected at the proximal part of the distal basal body that subtended the flagellar axoneme (Ax) close to the striated columns (SC, arrow). The schematic of the rat sperm head summarizes the observations from a series of images (schematic based on Fawcett, 1975). C, capitulum; IF, implantation fossa; Nuc, Nucleus. Scale bar, 200 nm.