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Fig. 5. LC3 is essential for starvation-induced degradation of p62. (A) HeLa cells were transfected with either transfection reagent (mock), nontargeting siRNA (control siRNA) or a pool of LC3 siRNAs (A, B and C isoforms) using DharmaFect reagent. After 72 hours, the cells were incubated in EBSS medium in the absence or presence of 0.1 µM Baf A, lysed with RIPA extraction buffer and analyzed by western blot with anti-p62, anti-actin and anti-LC3 antibodies. The asterisk indicates a nonspecific band. Arrows indicate LC3-I and LC3-II. (B) Quantification of relative p62 degradation (left panel) or relative p62 level (right panel) was performed as described in the Materials and Methods. Data are means ± s.d. of five independent experiments. The cells were transfected as in A, starved for 3 hours in the absence (C) or presence (D) of 0.1 µM Baf A, immunostained with anti-p62 and anti-LC3 antibodies, and analyzed using confocal microscopy. Scale bars: 5 µm.
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