Fig. 3.

Autophagosomal uptake of the proteasome depends on Ub conjugation (A) GFP-LC3B–overexpressing HeLa cells were transfected with control or anti-E1 (UBA1) siRNA oligonucleotides. After 2 d, cells were lysed, resolved via SDS/PAGE, and examined for E1 expression. (B) (i) After silencing of E1, cells were starved for amino acids for 4 h and then stained with anti-α6. (Scale bars: 10 μm.) White arrows point to proteasome-containing autophagosomes. (ii) Overlap coefficients of colocalization of the proteasome with LC3B-II were measured according to the method of Manders. *P < 0.000027. (C) HeLa cells were transfected with control or anti-E1 (UBA1) siRNAs. Cells were left untreated (Cont.) or starved to amino acids (Starv.) for the indicated times. Cell lysates were immunoprecipitated (IP) with anti-α6, and the precipitates were resolved, followed by immunoblotting (WB) with anti-LC3B and anti-α6.