Anti-LC3B antibody produced in rabbit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- Dilute primary Ab at -1:1000 and incubate at 4°C overnight.

Protocol tips
- Dilute primary Ab at -1:1000 and incubate at 4°C overnight.

Publication protocol

RLE-6TN cells were lysed in ice-cold RIPA lysis buffer (1 ml RIPA; 10 µl PMSF) for 30 min. Protein concentration was determined with a bicinchoninic acid protein assay kit and proteins (50 µg/lane) were separated by 12 and 15% SDS-PAGE and transferred to polyvinylidene fluoride membranes. Membranes were blocked at room temperature for 2 h with 5% skimmed milk in Tris-buffered saline with 0.1% Tween-20 (cat. no. ST825; Beyotime Institute of Biotechnology) and subsequently probed with the following antibodies at 4°C overnight: Anti-β-actin antibody (1:2,000; cat. no. bs-0061R; BIOSS; Beijing, China), Anti-HIF-1α (1:1,000; cat. no. 14179; CST Biological Reagents Co., Ltd.), anti-LC3I/II (1:1,000; cat. no. L7543; Sigma-Aldrich; Merck KGaA), anti-cleaved caspase-9 (1:300; cat. no. ab2325; Abcam), anti-caspase-8 (1:1,000; cat. no. ab25901; Abcam), anti-cleaved caspase-3 (1:1,000; cat. no. 9664; CST Biological Reagents Co., Ltd.). Following this, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000; ZB2301; OriGene Technologies, Inc.) antibody for 1 h at room temperature. Proteins were visualized with an enhanced chemiluminescence substrate kit (Clinx Science Instruments Co., Ltd., Shanghai, China) and standard X-ray film development (ChemiScope 3,300 mini; Clinx Science Instruments Co., Ltd.). Results were quantified with ImageJ software v1.48 (National Institutes of Health, Bethesda, MD, USA) and processed using Adobe Photoshop CS5 (Adobe Systems, Inc., San Jose, CA, USA).

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