Anti-LC3B antibody produced in rabbit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Homogenize cells in T-per buffer with a protease inhibitor cocktail.	- Boil samples in loading buffer before western blot analysis.

Upstream tips
- Homogenize cells in T-per buffer with a protease inhibitor cocktail.
Protocol tips
- Boil samples in loading buffer before western blot analysis.

Publication protocol

Autophagy determination
RFP-GFP-LC3- and GFP-LC3B -transfected cells were visualized by confocal microscopy. To quantify the number of puncta, RFP-GFP-LC3- or GFP-LC3B -transfected cells were seeded in a dish one day before the treatment. The cells were then analyzed by confocal microscopy. The number of puncta per cell was determined using Image-Pro plus 6.0. More than 10 cells were analyzed for each condition, and a cell was considered autophagic if more than 10 puncta were present in that cell.

Western blot analysis
Western blots were performed to determine the expression levels of LC3, p62 and β-actin. Briefly, AR42J cells were homogenized in T-per buffer (Thermo Fisher) with a protease inhibitor cocktail (Thermo Fisher). Lysates were centrifuged at 16,000 g for 15 min, and then, the samples were boiled in loading buffer before western blot analysis. The protein content was determined by the BCA assay. The western blot was performed according to standard procedures. The protein bands were detected by a ChemiDOC XRS imaging system (Bio-Rad, USA), and β-actin served as a loading control.

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Manufacturer protocol

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