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. 2023 Mar 2;222(4):e202206121. doi: 10.1083/jcb.202206121

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© 2023 Zhu et al.

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Figure 4. — Mmp9/Mmp14 regulates galectin-3 lattice remodeling in osteoclasts. (A and B) Galectin-3 expression and cleavage as assessed with anti–galectin-3 monoclonal antibody (ab2785; Abcam; epitopes mapped against N-terminal region) by Western blot (A) and quantification (B) in BMDMs and osteoclasts (OC) generated from wild-type or DKO mice. Data are presented as mean ± SEM (n = 3 biological replicates). (C and D) Galectin-3 (green) immunofluorescence of non-permeabilized wild-type or DKO osteoclasts stained with an anti–galectin-3 monoclonal antibody (#125401; Biolegend; clone M3/38, epitopes mapped against N-terminal region; C), and surface galectin-3 level quantified (D). Scale bar, 20 μm. Data are presented as mean ± SEM (n = 3 biological replicates with two technical replicates each). (E) Following cell surface biotinylation and capture with streptavidin magnetic beads, Mmp9, Mmp14, galectin-3, and β3 integrin expression in the membrane fraction of wild-type and DKO osteoclasts as assessed by Western blot. Results are representative of three independent experiments. (F) Measurements of surface galectin-3 in wild-type and DKO osteoclasts with eFluor 660–conjugated anti–galectin-3 monoclonal antibody (#50-5301-82; Thermo Fisher Scientific; clone M3/38, epitopes mapped within the N-terminal domain) by flow cytometry. Results are representative of three independent experiments. (G) Measurements of surface galectin-1 in wild-type and DKO osteoclasts by flow cytometry. Results are representative of three independent experiments. (H) DKO BMDMs were transduced with lentiviral vectors expressing full-length MMP9, an MMP9E/A mutant, or an empty control, and differentiated into osteoclasts. Cell lysates were collected for MMP9 and galectin-3 expression and cleavage as assessed by Western blot. Results are representative of three independent experiments. (I) DKO BMDMs were transduced with lentiviral vectors expressing full-length MMP14, MMP14E/A, or an empty control and differentiated into osteoclasts. Cell lysates were collected for MMP14 and galectin-3 expression and cleavage as assessed by Western blot. Results are representative of three independent experiments. **P < 0.01. Statistical significance was assessed using two-way ANOVA with Bonferroni correction (B) and unpaired two-sided Student’s t test (D). Source data are available for this figure: SourceData F4.