Fig. 3: Sod1 is secreted in an autophagy-dependent manner.
From: Plekhg5 controls the unconventional secretion of Sod1 by presynaptic secretory autophagy
Western blots of lysates and media from NSC34 cells treated with 3-MA (A), and GW4869 (B) for 8 h. C Quantification of the Sod1 intensities upon exposure to 3-MA or GW4869. n = 3; biological replicates. One-Sample t-test, two-tailed. D Western blot of the lysate and medium from NSC34 cells treated for 1 h with HBSS. The images are representative of four biological replicates. E Western blot showing similar LC3-II/ LC3-I ratios upon knockdown of Atg9 in MNs (sh-RNA-1= #1; sh-RNA-2 = #2). F Quantification of the LC3-II/LC3-I ratios normalized to the control. n = 4 biological replicates. Repeated measures one-way ANOVA. Dunnett’s multiple comparisons test. G Western blots showing reduced Sod1 levels in the medium of MNs upon knockdown of Atg9 in MNs. H Quantification of the Sod1 intensities. Lysate n = 4, medium n = 3 (sh-Atg9#1); Lysate n = 4, medium n = 4 (sh-Atg9#2); biological replicates. One-sample t-test, two-tailed. I Western blot showing a reduced LC3-II/LC3-I ratio in Atg5 depleted MNs. J Quantification of the LC3-II/LC3-I ratios normalized to the control. n = 4 biological replicates. t-test, two-tailed. K Depletion of Atg5 in MNs results in a reduced secretion of Sod1 as shown by Western blot. L Quantification of the Sod1 intensities. n = 4 biological replicates. unpaired t-test, two-tailed. M Membrane fractionation scheme. N–P Western blots showing the distribution of Sod1 and the indicated membrane markers in the different membrane fractions. T, top; B, bottom. The images are representative of two biological replicates. Q LC3-positive membranes were immunoisolated and the presence of Sod1 was determined by Western blot. FT flowthrough, IP immunoprecipitation. The images are representative of two biological replicates. Quantification of Sod1 secretion was calculated as the ratio between the amount of Sod1 in the medium and in the lysate. Sod1 levels in lysates were adjusted to Actin. The normalized Sod1 intensities were set to 1 in each experiment Data are mean ± SEM. Source data are provided as a Source Data file.
