LC3B Antibody Kit for Autophagy

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Chloroquine and Rapamycin treated cells are used as positive control	1:250 dilution of Primary antibody

Upstream tips
Chloroquine and Rapamycin treated cells are used as positive control
Protocol tips
1:250 dilution of Primary antibody

Publication protocol

Western blotting
Protein lysates from cells and flies were obtained by lysis in RIPA buffer (Sigma) at the indicated time points. Micro BCA protein kit (Pierce) was used to measure protein levels and equal amounts of protein were electrophoresed on 4–20% Tris-HCL Criterion gels (Biorad), and transferred onto nitrocellulose membranes. Western blots were analyzed using an Odyssey Imager (Licor) according to manufacturer’s instructions. Western blot figures are a representative of at least two independent experiments.

Immunofluorescence
Infected cells were immunostained and visualized as previously described3. Briefly, macrophages were seeded onto poly-lysine coated coverslips and infected with M. tuberculosis as described above. Cells were infected at an MOI of 1, and fixed in 4%PFA for 20 min at the indicated time points. Cells were incubated with indicated primary antibodies for 2 h at room temperature in 5% milk, 0.05% saponin and visualized using secondary Alexa-fluor488 antibodies. For Parkin immunofluorescence, cells were stained using rabbit polyclonal anti-Parkin (Abcam15954) and an HRP conjugated donkey anti-rabbit secondary (1:100, Jackson Immunochemicals) followed by amplification with alexafluor-488 tyramide (Invitrogen). Colocalization studies were performed as blinded experiments, with a minimum count of 200 cells per coverslip and performed in triplicate. Data shown is the mean ± SEM of at least 3 experiments.

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