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. 2010 Apr 23;29(11):1792–1802. doi: 10.1038/emboj.2010.74

Figure 2.

Figure 2

The GABARAP/GATE-16 protein subfamily is essential for autophagy. (A) HeLa cells stably expressing GFP-GATE-16 (left panel) or GFP-LC3B (right panel) were transfected with either non-targeting siRNA (control siRNA) or a pool of GABARAP siRNAs (GABARAP, GABARAPL1, and GATE-16) using DharmaFect reagent. After 72 h interval, the cells were incubated for 2 h in EBSS medium in the absence or presence of 0.1 μM Baf A and subjected to western blot analysis after lysis with RIPA extraction buffer. (B) Quantification of relative p62 and GFP-LC3B level (upper panel) and degradation (lower panel) were performed as described in ‘Materials and methods'. Mean±s.d. of three independent experiments is presented. (C) HeLa cells stably expressing GFP-LC3B were transfected with non-targeting siRNA or a pool of GABARAP siRNAs and treated as in (A). The cells were subjected to immunostaining with anti-p62 after fixation, and analysed by confocal microscopy. The GFP intensity of three independent experiments was measured as described in ‘Materials and methods' (right panel). Scale bar: 20 μm.