Anti-LC3B antibody (ab48394)

Protocol tips

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	- 1:500 dilution of primary antibody and overnight incubation at 4°C

Protocol tips
- 1:500 dilution of primary antibody and overnight incubation at 4°C

Publication protocol

To determine APN, LC3B, JNK/P-JNK, Akt/P-Akt, P62, and cleaved-caspase-3/pro-caspase-3 levels, proteins were extracted from the cells by suspension in radioimmunoprecipitation assay buffer. Samples were centrifuged at 12,000 rpm at 4°C for 30 min, and the supernatants were recovered for analysis. The protein concentrations were determined using the Bradford protein method and the bicinchoninic acid protein assay kit (Sigma, St Louis, MO, USA). Protein (40 µg) was electrophoresed on a pre-cast bis-Tris polyacrylamide gel (8%~12%) and then transferred onto a polyvinylidene difluoride membrane. Membranes were blotted with rabbit anti-APN (1:1,000), rabbit anti-LC3B (1:500), rabbit anti-JNK (1:1,000), rabbit anti-p-JNK (1:1,000), rabbit anti-Akt (1:1,000), rabbit anti-p-Akt (1:1,000), mouse anti-P62 (1:1,000), rabbit anti-Caspase-3 (1:1,000) (all from Abcam, San Francisco, CA, USA), and mouse anti-actin (1:1,000; Proteintech, NY, USA), followed by horseradish peroxidase-conjugated secondary antibodies (1:5,000; ZsBio, Beijing, China). Immunoblots were visualized using enhanced chemiluminescence (LAS-4000).

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Manufacturer protocol

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