Fig. 4

EndoA2 interacts with Piezo2 and regulates Piezo2 trafficking in sensory neurons. a–d Co-immunoprecipitation (Co-IP) results showed that EndoA2 potentially interacted with Piezo2 in DRGs. DRG lysates were immunoprecipitated with EndoA2 antibody and immunoblotted with Piezo2, TACAN, ASIC2, ASIC3 and EndoA2 antibody as indicated. This experiment was repeated 3 times. High-resolution images showing the colocalization of EndoA2 and Piezo2 in DRG neurons (e) and sciatic nerves (f) of mice. Arrows indicate colocalization. Scale bar = 5 μm. g Proximity ligation assay (PLA) showed positive signals of EndoA2/Piezo2 interaction in cultured DRG neurons, and staining was absent with incubation of EndoA2 and ASIC2 antibody (6 images from two repeats). Scale bar = 30 μm. h Diagram of full-length EndoA2 and EndoA2 mutant proteins. i Interaction between Piezo2 and EndoA2 mutants. The EndoA2 mutants were transiently coexpressed with Piezo2, and the cell lysates were immunoprecipitated with antibodies as indicated. This experiment was repeated 3 times. j–l Piezo2 expression in the DRG neuron membrane fraction (j), cytoplasmic fraction (k) and total lysate (l) from EndoA2^fl/fl, EndoA2^fl/fl × Nefh-Cre and rescue mice. n = 4–5. m Statistical diagram of Piezo2 expression in the DRG neuron membrane fraction (j), cytoplasmic fraction (k) and total lysate (l) from EndoA2^fl/fl, EndoA2^fl/fl × Nefh-Cre and rescue mice. n = 4–5. n–p Piezo2 surface levels were measured in cultured DRG neurons prepared from EndoA2^fl/fl, EndoA2^fl/fl × Nefh-Cre and rescue mice using a surface biotinylation assay. n = 3 repeats per group. One-way ANOVA followed by Tukey’s multiple comparisons test (m, n, o, p). ^**P < 0.01, ^***P < 0.001, ns non-significant. The error bars indicate the SEMs. EndoA2 endophilin A2, DRG dorsal root ganglion, PLA proximity ligation assay, TfR transferrin receptor