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. 2023 Feb 4;14:616. doi: 10.1038/s41467-023-36168-x

Fig. 5. La associates with the surface of osteoclasts by direct interactions with Anx A5.

Fig. 5

a Immunoprecipitation of osteoclast lysates 3 days post-RANKL addition. La supermolecular complexes were captured on immunomagnetic beads via α-La mAb or isotype control and complexes were blotted with rabbit antibodies raised towards the targets of interest (α-La rAb for La). Lanes from the same blot are presented at the same intensity. Lanes of interest were cropped and placed beside one another, divided by a dashed line. (b) Representative Western blot of magnetic, streptavidin pull-down of Biotin-Anx A5. Lane 2 = La+Anx A5 input before pull-down and Lane 1 = La alone and Lane 3 = La+Anx A5 after pull-down. c A cartoon illustration of our approach to identify membrane affinity by comparing protein contents in the Bottom fraction containing, along with soluble proteins, liposome-bound proteins and in the Top fraction containing soluble proteins and depleted of liposome-bound proteins. (d) Quantification of the enrichment of recombinant La and Anx A5 in the bottom fraction containing pelleted liposomes (n = 2). (e) Quantification of surface fluorescence intensity of Anx A5 or La following either non-targeted or Anx A5-targeted siRNA in human osteoclasts (n = 4) (P = 0.009 and 0.004, respectively). (f) Quantification of fusion events from e (n = 3) (P = 0.007). (g) Representative immunofluorescence images of Anx A5 or La (α-La mAb) surface staining in non-permeabilized, human osteoclasts 3 days after RANKL addition before or after EGTA incubation. (h) Quantification of surface fluorescence intensity from g. (Anx A5 and RANK n = 2; La n = 3) (P = 0.02, 0.04 and 0.3, respectively) (i) Quantification of surface fluorescence intensity of Anx A5, La or RANK treated or not treated with 60 μM A01 (n = 3) (P = 0.006, 0.01 and 0.35, respectively). In e, h and I, ~100 cells were assessed per each condition in each independent experiment. (d, e, f, h, i). Data are presented as mean values + /- SEM. Statistical significance was assessed via one-tailed paired t-test. * = P < 0.05; ** = P < 0.001. Source data are provided as a Source Data file.