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Fig. 6. Knockdown of LC3 leads to accumulation of detergent-insoluble p62 and polyubiquitylated proteins. (A) HeLa cells were transfected with either nontargeting siRNA (control siRNA), or with a pool of LC3 siRNAs (A, B and C isoforms) using DharmaFect reagent. After 72 hours, the cells were incubated in EBSS medium, lysed with RIPA extraction buffer and analyzed by western blot with anti-p62, anti-actin and anti-ubiquitin antibodies (left panel). Quantification of relative ubiquitin level (right panel) was performed as described in Materials and Methods. Mean ± s.d. of three independent experiments is presented below. (B) The cells were treated as in A, fixed, immunostained using anti-LC3, anti-ubiquitin (UB) and anti-p62 antibodies, and analyzed using confocal microscopy. (C) Cells were transfected as in A, starved for 3 hours in the presence of Baf A, then treated for 20 minutes with 1% Triton X-100 (right panel), fixed and stained with anti-LC3, anti-ubiquitin (UB) and anti-p62 antibodies. Scale bars: 5 µm. (D) The cells were transfected as in A, starved for 3 hours, lysed with RIPA extraction buffer, the lysates were centrifuged for 30 minutes at 350,000 g. The supernatant (soluble, left) and pellet (insoluble, right) fractions were analyzed by western blot with anti-p62, anti-actin and anti-ubiquitin antibodies.
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