Fig. 4: Loss of DNRX leads to abnormal accumulation of DG in vivo and in vitro.

a–d Immunostaining revealed DG localization in wild-type and dnrx²⁷³ mutants backgrounds. An anti-HA antibody was used to label DG (red), and DAPI (blue) was used to label the nucleus. Scale bars, 50 μm (a, b) and 5 μm (c, d). e, f ’ Immunostaining revealed that DG was localized on the muscle cell membrane in the wild-type and dnrx²⁷³ mutants backgrounds. An anti-HA antibody was used to label DG (red), and DLG (green) represents the postsynaptic muscle membrane. Scale bar, 10 μm. g Quantification of the fluorescence intensity ratio (HA/DLG) in the wild-type flies and dnrx²⁷³ mutant backgrounds. The data are presented as the means ± SEMs; mef2-Gal4 > UAS-Dg-HA, n = 9; mef-Gal42 > UAS-Dg-HA, dnrx²⁷³, n = 8. *p < 0.05; two-tailed unpaired Student’s t-test. h Western blotting revealed an increase in DG levels in the muscle of dnrx²⁷³ mutants. An anti-HA antibody used to label DG. The protein originated from larval body wall muscles. i Quantification of DG protein levels relative to tubulin levels. The data are presented as the means ± SDs of three independent experiments. *p < 0.05; two-tailed unpaired Student’s t-test. j Quantification of the mRNA levels of mef2 in the muscles of w¹¹¹⁸ flies and dnrx²⁷³ mutants. The data are presented as the means ± SDs of three independent experiments. ns, p > 0.05; two-tailed unpaired Student’s t-test. k, k” Immunofluorescence images of HEK293T cells transfected with DG alone. l–l” Immunofluorescence images of HEK293T cells transfected with DG and DNRX^FL. m–m” Immunofluorescence images of HEK293T cells transfected with DG and DNRX^ΔCyto. n–n” Immunofluorescence images of HEK293T cells transfected with DG and DNRX^ΔExtra. DAPI was used to stain the nucleus. Scale bar, 5 μm. k”’, l”’, m”’, n”’ Plots of pixel intensity along the straight dashed white line in the images to the left of each plot. p: Pearson’s coefficient.