Anti-LC3B antibody produced in rabbit

Protocol tips

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	For WB, dilute primary antibody (1:1000) overnight at 4 °C

Protocol tips
For WB, dilute primary antibody (1:1000) overnight at 4 °C

Publication protocol

FP-LC3 transfection and fluorescence microscopy
The GFP-LC3 plasmids were transfected into HeLa cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s guidelines. The formation of GFP-LC3 puncta was observed under a fluorescence microscope (DMI 3000B, Leica) after cells were treated with the rapamycin and HVJ-E as indicated. Cells with five or more puncta were considered to have accumulated autophagosomes, because up to four puncta were observed in a small number of untreated cells. A total of 100 transfected cells were analyzed in each well, and three independent experiments were performed.

Western blot analysis
HeLa cells were treated with HVJ-E for 24 h. Proteins were extracted from infected and non-infected cells using a cell lysis buffer. Proteins were then separated by 8–15% SDS-PAGE and transferred to a PVDF membrane. The membranes were blocked in 5% non-fat milk for 1 h before incubation with primary antibody (1:1000) overnight at 4 °C. The bound antibody complexes were detected using a chemiluminescence reagent after membranes were incubated with HRP-conjugated IgG secondary antibodies (1:5000).

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