Western blot analysis of activation of caspases in A549 cells
This protocol is extracted from research article:
A self-illuminating nanoparticle for inflammation imaging and cancer therapy
Sci Adv, Jan 9, 2019; DOI: 10.1126/sciadv.aat2953

A549 cells were seeded into a six-well plate at 3 × 105 cells per well. After 12 hours, cells were treated with CLP nanoparticles at doses of 0, 65, or 173 μM for 24 hours. Then, cells were washed three times with ice-cold PBS and lysed in ice-cold radioimmunoprecipitation assay buffer containing protease inhibitors. The supernatant was collected by centrifugation at 12,000g for 5 min at 4°C. The concentration of total protein was quantified by the bicinchoninic acid (BCA) method. The protein samples were loaded into SDS–polyacrylamide gel electrophoresis. The separated proteins were transferred to polyvinylidene fluoride membranes, followed by blocking in tris-buffered saline–Tween 20 (TBST) containing 5% nonfat dry milk at room temperature for 2 hours. Subsequently, each membrane was incubated at 4°C overnight with respective primary antibodies. Membranes were washed three times with TBST, followed by incubation with appropriate horseradish peroxidase–labeled antibodies at room temperature for 1 hour. Last, membranes were washed and then detected using chemiluminescence reagents.

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