LC3B (D11) XP® Rabbit mAb (Biotinylated)

Autophagy assay cell type - Beas-2B

Experiment
Autophagy assay cell type - Beas-2B
Product
LC3B (D11) XP® Rabbit mAb (Biotinylated) from Cell Signaling Technology
Manufacturer
Cell Signaling Technology

Protocol tips

Upstream tips
- Lyse cells in RIPA buffer (2 mM pH 8.0 ETDA, 50 mMTris pH 7.4, 150 mMNaCl, 1% NP-40, 1 mM PMSF, 1% sodium deoxycholate) containing protease inhibitor cocktail
Protocol tips
- Dilute Ab at 1:1000 and incubate overnight at 4°C with primary antibody

Publication protocol

Following treatment protocols, cells were washed twice with ice-cold PBS (2.7 mMKCl, 2 mM KH2PO4, 137 mMNaCl, 10 mM Na2HPO4, pH 7.4), then lysed with RIPA buffer (2 mM pH 8.0 ETDA, 50 mMTris pH 7.4, 150 mMNaCl, 1% NP-40, 1 mM PMSF, 1% sodium deoxycholate) containing protease inhibitor cocktail (Roche, 04693116001). The concentration of protein extracts was assayed with Bio-Rad Protein Assay Kit (Bio-Rad, 500-0006). Cell lysates were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to hydrophobic polyvinylidene fluoride transfer membrane (PVDF) (PALL, 29301-808). Membranes were blocked in TBS containing 5% non-fat milk and 0.2% Tween 20 for 1 h and probed overnight at 4°C with primary antibodies. Antibodies against HK2 (2867), PARP (9542), LC3B (3868), cleaved caspase-7 (9491), cleaved caspase-3 (9661), p-AKTSer473 (9271) and p-AMPKαThr172 (2535) were obtained from Cell Signaling Technology. Antibody against p-p70S6kThr389 (MABS82) was purchased from Millipore. Antibody against LCN2 (AF1757) was supplied by R&D Systems and β-actin (A5441) was supplied by Sigma. Blots were washed in 0.2% TBS-Tween 20 and incubated with HRP-conjugated secondary antibodies for 1 h. The protein blots were visualized using enhanced chemiluminescence reagent obtained from Perkin Elmer (NEL105001EA).

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Papers

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Paper title
Metformin alleviates nickel-induced autophagy and apoptosis via inhibition of hexokinase-2, activating lipocalin-2, in human bronchial epithelial cells
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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for LC3B (D11) XP® Rabbit mAb (Biotinylated) below.

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