|Dilute primary Ab at 1:1000 and incubate overnight at 4°C
The protein content of the cellular lysates was determined using Bicinchoninic Acid (BCA) method. 20–40 μg of the protein samples were loaded on SDS-PAGE gel and electrophoresed. Electrophoresed proteins were transferred on to the Polyvinylidene difluoride (PVDF) membrane using wet transfer for 2.5 hrs at 60 V. The membrane was blocked for 1 hr at RT with 5% w/v non-fat dry milk in 1 X TBST (Tris-buffered saline with 0.1% Tween-20). Subsequently, membrane was incubated overnight at 4°C with anti-LC3 primary antibody (Cat. No. #12741, obtained from Cell Signalling Technologies, dilution-1:1000) in 5% w/v Bovine Serum Albumin (BSA) in TBST. This was followed by incubation with anti-rabbit HRP antibody (Cat. No. #7074, procured from Cell Signalling Technologies, dilution-1:5000) for 1 hr at RT. The proteins on the blot were then detected using chemiluminescence method (BioRad Clarity Western ECL substrate). LC3-II expression levels (which correlates to the number of autophagosomes formed) were then quantitated using Image-J software. Full paper
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