Fig. 4

RUFY4 acts as an adaptor between Rab7 and LAMP2. a–c Co-IP analysis of association of RUFY4 with either Rab7 (a), LAMP2 (b), or both (c) in HEK293T cells that were transfected to express the indicated proteins (n = 3). d Co-IP analysis of endogenous association of Rab7 and LAMP2 with transduced Flag-RUFY4 in mature osteoclasts (n = 3). e Co-IP analysis of endogenous association of Rab7 and LAMP2 in mature Rufy4^+/+ and Rufy4^−/− osteoclasts (n = 3). f Immunofluorescence staining of endogenous Rab7 and LAMP2 in HeLa cells that were transfected with EV-EGFP or RUFY4-EGFP. In EGFP-expressing cells, the percentage of Rab7⁺ LAMP2⁺ area among total vesicles or mean LAMP2 vesicle distance to nucleus was quantified (n = 3). g, h Co-IP analysis of association between ΔRUN or ΔFYVE mutants of RUFY4 with Rab7 (g) or LAMP2 (h) in HEK293T cells (n = 3). i Fluorescence staining of HeLa cells that were co-transfected with EV-EGFP, RUFY4-EGFP, or ΔFYVE-EGFP, and mCherry-2xFYVE. White arrows indicate the co-localization of RUFY4-EGFP with mCherry-2xFYVE, which labels PI(3)P-containing organelles (n = 3). Scale bars, 10 μm. j Co-IP analysis of association between ΔCC mutant of RUFY4 and full-length RUFY4 in HEK293T cells (n = 3). k, l Rufy4^−/− osteoclasts were transduced with full-length RUFY4 or its domain mutants and cultured on bone slices. k Hematoxylin staining was conducted to visualize resorption pits (n = 5). Scale bar, 100 μm. l Immunoblot analysis of active CTSK levels in the cell lysates and supernatants (n = 3). Actin served as a loading control. Western blots were probed with the indicated antibodies. Band intensity was determined with ImageJ. The data are presented as mean ± SD. Two-tailed unpaired Student’s t test (d–f) or one-way ANOVA with Tukey’s multiple comparison post hoc test (i, k, l) was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.000 1