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Detection of autophagic flux

AR Antonella Rigo
IF Isacco Ferrarini
EL Erika Lorenzetto
ED Elena Darra
IL Irene Liparulo
CB Christian Bergamini
CS Cinzia Sissa
EC Elisabetta Cavalieri
FV Fabrizio Vinante
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Jurkat cells were treated with vehicle or 80 μM BSB for 16 h. They were harvested, washed in cold PBS, and resuspended in Lysis Buffer (Thermo Fisher Scientific) plus protease inhibitor cocktail (Sigma-Aldrich). Whole-cell lysates were separated on a 12% SDS–PAGE and analyzed by immunoblotting as described above.

LC3: In order to inhibit the degradation of autophagic cargo, Pepstatin A (Sigma-Aldrich, 10 μg/mL) and E-64D (Sigma-Aldrich, 10 μg/mL) were added to the media. Membranes were tested with anti-LC3 (Cell Signaling Technology, no. 12741) and anti-GAPDH antibodies (Cell Signaling Technology, no. 2118).

Beclin 1: Membranes were tested with anti-Beclin 1 (Cell Signaling Technology, no. 3495) and anti-GAPDH antibodies. Control and BID-knockdown Hela cells were also tested.

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