Fig. 3. La associates with membranes, traffics to the surface and controls osteoclast membrane fusion.

a Westerns of cytosolic vs membrane associated protein fractions from human osteoclasts. b Representative immunofluorescence images comparing surface staining with α-Fish/TKS5 antibody or α-La mAb in human osteoclasts under non-permeabilized conditions (top) and DIC (bottom). c Representative immunofluorescence images comparing surface staining of isotype control or α-La mAb in RAW 264.7 derived osteoclasts under non-permeabilized conditions. (α-La mAb). d Representative immunofluorescence images of cell surface La in forming human osteoclasts 2–5 days post RANKL application. Cells were stained with α-La mAb at the described timepoints without membrane permeabilization. e Cartoons illustrating the stepwise process of the formation of multinucleated osteoclasts (top), and our approach for isolating membrane fusion stage from the preceding stages of osteoclast differentiation (bottom). Application of the hemifusion inhibitor LPC following 48 h of RANKL elicited osteoclastogenesis allows pre-fusion differentiation stages but blocks hemifusion, synchronizing cells. Removing LPC allows us to specifically probe membrane fusion between osteoclasts. f Quantification of human osteoclast fusion decoupled from pre-fusion stages and synchronized as depicted in e with fusion in the presence of 5 μg/ml α-La mAb and with no antibodies added (Wash) normalized to 5 μg/ml α-RANK control. (wash n = 2; others n = 4) (P = 0.0086 and 0.1330, respectively). “LPC” – fusion observed without removal of LPC. Statistical significance was assessed via one-tailed paired t-test. * = P < 0.05. ** = P < 0.001. Data are presented as mean values + /- SEM. Source data are provided as a Source Data file.