Figure 5.

Ifnb^–/– Neurons Have a Defect in Autophagy Maturation

(A–H) Ifnb^+/+ and Ifnb^–/– primary CNs cultured for 21 days.

(A) WB from three independent experiments with antibodies as indicated.

(B) Quantified IOD of WB bands normalized to vinculin. Data are mean ± SEM, n = 3.

(C) WB of CNs with or without NH₄Cl (20 mM) for 1 or 4 hr.

(D) Quantified IOD of WB. Data are mean ± SEM of n = 3, ^∗/^#p < 0.05, ^∗∗/^##p < 0.01 by one-way ANOVA. ^∗Within and # between genotype differences after NH₄Cl treatment.

(E) CNs expressing mRFP-GFP-LC3B with or without NH₄Cl for 2 hr. Arrowheads, colocalized GFP and mRFP (autophagosomes); arrows, mRFP-only vesicles (autolysosomes). Scale bars, 10 μm; 2 μm in inserts.

(F) Graphs, mean number of autophagosomes and autolysosomes/cell and vesicle ratio ± SEM, n = 3.

(G) WB of Rab7. Graph, IOD of WB. Data are mean ± SEM of n = 3.

(H) IF showing LC3B and p62 colocalized in autophagosomes (arrowheads) and LC3B, LAMP1, and p62 colocalizing in autolysosomes (arrows). Scale bars, 10 μm; 2 μm in inserts.

For (B), (F), and (G), ^∗p < 0.05, ^∗p < 0.01, ^∗∗∗p < 0.001 by unpaired Student’s t test. See also Figure S5.