The immunoblotting assay was performed according to previous study (Huang et al., 2015 (link)). Briefly, cells were lysed in RIPA buffer with protease inhibitor cocktail (Solarbio, Beijing, China). The bicinchoninic acid method was used to determine the protein content. Proteins were separated by SDS-PAGE gels before electroblotted to PVDF membranes (Millipore, Billerica, MA, United States). The membranes were blocked in 5% fat-free milk for 1 h, followed by incubation with primary antibodies against protein kinase B (AKT; Abcam, Cambridge, UK), p-AKT (Abcam), forkhead box O3 (FOXO3; Abcam), p-FOXO3 (Abcam), Beclin1 (Abcam), microtubule-associated protein light chain 3 (LC3; Cell Signaling Technology, Beverly, MA, United States), and GAPDH (Zhongshan Goldenbridge Biotechnology Co., Beijing, China) at 4°C overnight. The next day, the membranes were washed before incubation with secondary antibodies (Zhongshan Goldenbridge Biotechnology Co.) for 1 h. The blots were detected using an enhanced chemiluminescence substrate kit (Applygen, Beijing, China) and quantified using ImageJ software. The intensity of each signal was normalized to GAPDH values.
Microtubule associated protein light chain 3 lc3
Manufactured by Cell Signaling Technology
Sourced in United States
Microtubule-associated protein light chain 3 (LC3) is a protein involved in the formation of autophagosomes, which are double-membrane vesicles that deliver cytoplasmic cargo to the lysosome for degradation. LC3 is a commonly used marker for monitoring autophagy.
Automatically generated - may contain errors
Lab products found in correlation
P akt, by Abcam (1 mentions)
P foxo3, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Solarbio (1 mentions)
Beclin 1, by Abcam (1 mentions)
Gapdh, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Rapamycin rp, by Merck Group (1 mentions)
3 methyladenine 3 ma, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
3 protocols using microtubule associated protein light chain 3 lc3
1
Immunoblotting Assay Protocol for Protein Analysis
2
Western Blot Analysis of Autophagy Regulators
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Cells were harvested from the plates. After digestion and centrifugation, total cell proteins were extracted from the cells using RIPA lysis method. Then, 100 μg proteins were applied onto 10% SDS- polyacrylamide gel (SDS-PAGE) for electrophoresis and then transferred onto nitrocellulose membrane. After blocking for 2 h, members were incubated overnight at 4°C with the antibodies (CSN6, 1:2 000; mTOR, 1:500 dilution, Proteintech Group, USA; Microtubule-associated protein light-chain 3 (LC3), 1:2 000 dilution, Cell Signaling Technology, USA; Sequestosome 1 (SQSTM1/p62), 1:2 000 dilution, Cell Signaling Technology, USA; CTSL, 1:300 dilution, Santa Cruz Biotechnology, USA; β-actin, 1:2 000 dilution, Zhong-shan biotech, China). After washed, the secondary antibody was added to incubate at room temperature for 2 h prior to ECL (Tanon, Shanghai, China) fuorescence imaging.
3
Autophagy Regulation in QBC939 Cells
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The established QBC939 cell line was obtained from the Cell Bank of Wuhan University (Wuhan, China). OC, 3-methyladenine (3-MA) and rapamycin (RP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). β-actin, microtubule-associated protein light chain 3 (LC3) and p62 antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). All other chemicals were of analytical grade.
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