Figure 2.

Reduced interaction between Beclin 1, TAB and TAB3 in conditions of autophagy induction. (A, B) Inhibition of autophagy by dominant-negative (DN) TAK1. HeLa cells were co-transfected with a GFP–LC3-encoding construct plus pcDNA3.1 (empty vector), or plasmids for the expression of WT TAK1 (TAK1^WT) or the DN TAK1^K63W mutant. One day later, cells were either left untreated (control) or driven into autophagy by starvation or by the administration of 1 μM rapamycin or 30 μM pifithrin α (PFTα), followed by immunoblotting for the detection of TAK1 and endogenous LC3 (A) or immunofluorescence microscopy for the quantification of cells with cytosolic GFP–LC3 puncta (GFP–LC3^VAC cells) (B) (mean values±s.d., n=3; ^*P<0.01 versus control cells). GAPDH levels were monitored to ensure equal loading. (C, D) Inhibition of autophagy by knockdown of VPS34, Beclin 1 (BCN1) and TAK1. siRNAs that effectively deplete VPS34, BCN1 and TAK1 were co-transfected with a GFP–LC3-encoding plasmid in HeLa cells. Autophagy was then induced as in (A) and the frequency of GFP–LC3^VAC cells (mean values±s.d., n=3; ^*P<0.01 versus control cells) was determined. (E, F) The same setting shown in (C, D) was performed with U2OS cells and FYVE–RFP (mean values±s.d., n=3; ^*P<0.01, ^**P<0.001 versus control cells).