Anti-LC3B antibody produced in rabbit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Lyse cells on ice with RIPA buffer	- Dilute Primary Ab -1:1000 dilution and incubate at 4°C overnight

Upstream tips
- Lyse cells on ice with RIPA buffer
Protocol tips
- Dilute Primary Ab -1:1000 dilution and incubate at 4°C overnight

Publication protocol

SDS-PAGE and immunoblotting were performed according to standard procedures to detect the expression levels of autophagy marker proteins, such as LC3. Briefly, cells were lysed with RIPA buffer on ice, and supernatant samples were resolved in 10–15% SDS-PAGE depending on the size of the target protein, followed by transfer onto nitrocellulose membranes (Millipore, Billerica, MA, USA). Thereafter, membranes were incubated in blocking buffer at room temperature for 1 h, followed by incubation with anti-LC3 antibody at 4°C overnight. The protein-antibody complexes were then tagged with IR Dye 680-labeled secondary antibodies at room temperature for 1 h. The infrared fluorescence was detected with the Odyssey infrared imaging system (Li-Cor Bioscience, Lincoln, NE, USA). β-actin was used as a loading control. The ratios of LC3-II/LC3-I to β-actin in each experiment were calculated by Quantity One software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

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