Anti-LC3 pAb

Protocol tips

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	Dilute at 1:2000 and incubate overnight at 4 °C

Protocol tips
Dilute at 1:2000 and incubate overnight at 4 °C

Publication protocol

7.5 × 105 HEK-293 cells were passaged on plates (6 cm in diameter), and allowed to attach overnight. Cells were then transfected, and treated with DMSO (control cells) or 30, 60 or 100 µM genistein for 48 h at 37 °C. In samples with lysosome inhibition, chloroquine was added to final concentration of 10 μM for 1 h before the genistein/DMSO treatment. Next, cells were lysed with a solution containing 1% Triton X-100, 0.5 mM EDTA, 150 mM NaCl, 50 mM Tris, pH 7.5, and a mixture of protease and phosphatase inhibitors (Roche Applied Science, #05892791001 and #11873580001). The lysates were cleared by centrifugation; proteins were separated by SDS-PAGE and transferred to a PVDF membrane overnight. The membrane was blocked with 5% non-fat dry milk in PBST buffer and then incubated with primary antibodies overnight at 4 °C. At the next day, the membrane was incubated with secondary antibodies at room temperature for 1 h, treated with a solution of substrates for detecting of HRP, and exposed to the X-ray film. The intensities of bands were analyzed with the QuantityOne software. Since aggregates of huntingtin do not enter the separating gel but they remain on top of the lane in the stacking gel, they were detected after transferring the material from the latter gel onto the PVDF membrane. Soluble huntingtin was analyzed from the separating gel. To quantify the amounts of both forms of huntingtin, β-actin was used as an internal control, and the results were normalized relative to the β-actin level.

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