Fig. 7

KIF5B is involved in EndoA2-mediated Piezo2 membrane trafficking. a Co-IP showing the EndoA2/KIF interaction in the DRG. DRG lysates were immunoprecipitated with an EndoA2 antibody and immunoblotted with KIF5B, KIF5A, KIF17, KIF3A, KIF3B and EndoA2 antibodies as indicated. This experiment was repeated 3 times. b Proximity ligation assay (PLA) shows positive signals of EndoA2/KIF5B interaction in cultured DRG neurons, and staining was absent with incubation of EndoA2 and KIF17 antibody (7 images from two repeats). Scale bar = 15 μm. c Interaction between KIF5B and EndoA2 mutants. The EndoA2 mutants were transiently coexpressed with KIF5B, and the cell lysates were immunoprecipitated with antibodies as indicated. This experiment was repeated 3 times. d The interaction level between Piezo2 and KIF5B was examined by Co-IP in EndoA2^fl/fl and EndoA2^fl/fl × Nefh-Cre mouse DRG lysates. DRG lysates were immunoprecipitated with Piezo2 antibody and immunoblotted with KIF5B and Piezo2 antibody as indicated. This experiment was repeated 3 times. e PLA shows positive signals of KIF5B/Piezo2 interaction in cultured DRG neurons of EndoA2^fl/fl mice, and staining was absent in cultured DRG neurons of EndoA2^fl/fl × Nefh-Cre mice (5 images from two repeats). Scale bar = 15 μm. f High-resolution images show the colocalization of KIF5B, EndoA2 and Piezo2 in DRG neurons. Arrows indicate colocalization. Scale bar = 5 μm. g Changes in the membrane expression of Piezo2 after KIF5B knockdown in the DRG. n = 6. The behaviors of NT-shRNA- and KIF5B-shRNA-injected mice were evaluated by cotton (h), foot tape (i) and 0.16 g punctate (j) tests. The same groups of mice were used for each pain behavioral test. The inter-test interval was at least 2 h. n = 6. k–n The effects of KIF5B knockdown on the punctate and dynamic mechanical allodynia induced by SNL and CFA. n = 8. o EndoA2 knockdown inhibited the increased expression of Piezo2 in the membrane of DRGs induced by KIF5B overexpression. n = 4–5. p KIF5B knockdown repressed the increased expression of Piezo2 in the membrane of DRGs induced by EndoA2 overexpression. n = 4–5. Two-tailed independent Student’s t test (g–j); Two-way ANOVA followed by Bonferroni’s multiple comparisons test (k–n); One-way ANOVA followed by Tukey’s multiple comparisons test (o, p). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ns non-significant. The error bars indicate the SEMs. EndoA2 endophilin A2, DRG dorsal root ganglion, TfR transferrin receptor, NT nontargeting, SNL spinal nerve ligation, CFA complete Freund’s adjuvant