Anti-LC3-I/II Antibody

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	1:1000 dilution of Primary antibody

Protocol tips
1:1000 dilution of Primary antibody

Publication protocol

U373MG cells were collected and washed twice with cold PBS after treatment with various quercetin concentrations. The cells were then lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA, 1 mM EGTA, 1 mM NaVO3, 10 mM NaF, 1 mM DTT, 1 mM PMSF, 25 μg/mL aprotinin, and 25 μg/mL leupeptin) and kept in ice for 30 min. The lysates were centrifuged for 30 min at 13,000 rpm and 4°C, and the supernatants were stored at –70°C until use. Cytosolic and mitochondrial extracts were prepared using fraction lysis buffer (75 mM NaCl, 8 mM Na2HPO4, 1 mM Na2H2PO4, 250 mM sucrose, 1 mM EDTA, and 350 μg/mL digitonin). Lysed cells were kept in ice for 10 min and then centrifuged for 15 min at 15,000 rpm and 4°C. The supernatant was the cytosolic fraction. After the pellet was washed with lysis buffer, it was lysed in lysis buffer to prepare whole lysates. Protein concentration was measured using a BCA Protein Assay kit. Aliquots of the lysates (30–70 μg protein) were separated via 10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a PVDF membrane using glycine transfer buffer (192 mM glycine, 25 mM Tris-HCl, pH 8.8, and 20% methanol, v/v). After blocking with 5% nonfat dried milk, the membranes were incubated for 4 h with primary antibodies, followed by an additional 30 min incubation with secondary antibodies in milk containing Tris-buffered saline and 0.1% Tween 20. Human anti-caspase-3, -caspase-7, -caspase-8, cleaved PARP, cytochrome c, JNK, phospho-JNK, p53, HSP60, LC3II, and Beclin-1 antibodies were used at a 1 : 1,000 dilution as the primary antibodies, and horseradish peroxidase-conjugated goat antihuman IgG was utilized as the secondary antibody at a 1 : 5,000 dilution. The membranes were then exposed to X-ray film. Protein bands were detected using the WEST-ZOL plus Western blot detection system (Intron, Gyeonggi-do, Republic of Korea).

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