LC3A/B Antibody

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	Dilute primary Ab at 1:300

Protocol tips
Dilute primary Ab at 1:300

Publication protocol

MCF7 cells were irradiated for 24 h (a time selected based on a time-course curve) and harvested; 1 ml lysis buffer (50 mM NaCl, 10 mM HEPES, 1 mM EDTA, 1 mM DTT, 1% NP-40, 0.1% SDS and 1 mM PMSF) and 10 µl protease inhibitor cocktail (P8340; Sigma-Aldrich; Merck KGaA) was added to the cell pellets. Subsequently, protein was quantified using BCA Protein Assay kit (P0012S; Beyotime Institute of Biotechnology, Haimen, China). SDS-PAGE was used to separate 60 µg of total cellular protein extract; the separated bands were transferred onto a nitrocellulose membrane for western blotting. Membranes were blocked with 5% skimmed milk in Tris-Buffered Saline Tween-20 for 1 h at room temperature. Subsequently, horseradish peroxidase-conjugated secondary antibodies [HRP-Goat anti-Rabbit IgG (cat no. bs-0295G-HRP) and HRP-Goat anti-Mouse IgG (cat no. bs-0296G-HRP; both 1:2,000); Boster Biological Technology, Pleasanton, CA, USA] was added for 1 h at room temperature. Primasry antibodies were used against DRAM1 (1:300; ab72171; Abcam; Cambridge, UK), light chain 3 (LC3) I and II (1:300; 4108S; Cell Signaling Technology; Danvers, MA, USA), and GAPDH (1:1,000; sc-32233; Santa Cruz Biotechnology, Inc.). Immunoreactive bands were detected by chemiluminescence (sc-2048; Santa Cruz Biotechnology, Inc.). The staining intensities of individual proteins were measured using ImageJ software (version 1.41; National Institutes of Health, Bethesda, MD, USA), and expressed relativeto the intensity of the GAPDH band.

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Manufacturer protocol

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