Fig. 3.

UBR4 plays a role in delivery of autophagic cargoes to phagophores, and UBR4 subpopulation is degraded by autophagy through starvation-enhanced association with autophagic core machinery. (A–C) Costaining of UBR4 in comparison with LC3 (A), ATG5 (B), and ATG12 (C) in HEK293–UBR4V5 cells. (D–F) Enlarged views of areas indicated by rectangles in A–C. (G and H) EM of HEK293–UBR4V5 cells labeled for UBR4 (12 nm) and ATG12 (18 nm). (Scale bar, 200 nm.) (I–K) PLA assay of UBR4 in comparison with ATG12 (I), ATG5 (J), and LC3 (K) in HEK293–UBR4V5 cells. (Scale bar, 1 μm.) (L) Immunoblotting of +/+ (WT) and UBR4^−/− (KO) MEFs growing in normal or starvation medium for 90 min in the presence or absence of 0.2 μM bafilomycin A1 or 10 mM 3-methyladenine. (M) Immunoblotting of MEFs cultured in normal media or HBSS for 90 min, with or without being supplemented by 10% (vol/vol) FBS. (N) Cyclohexamide-chase assay of UBR4 in MEFs. Cells were cultured in normal or starvation medium for 6 h in the presence of cycloheximide. Shown is a quantitation of a representative blot image from three independent experiments.