Anti-Beclin 1 antibody [2A4] (ab114071)

Protocol tips

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	Dilute primary Abbetween 1:500- 1:2000

Protocol tips
Dilute primary Abbetween 1:500- 1:2000

Publication protocol

Caspase 1 inhibitor (Ac-YVAD-CMK) was from Millipore. Antibodies for Western blot analysis were as follows: rabbit anti-caspase 1 was from Millipore; mouse anti-GAPDH was from Abcam; rabbit anti-beclin 1 was from Abcam and Cell Signaling Technology, Inc.; rabbit anti-cleaved caspase 3, cleaved poly(ADP-ribose) polymerase (PARP), caspase 3, PARP, Atg3, Atg12-Atg5 conjugate, and Atg7 were from Cell Signaling Technology, Inc.; and mouse anti-cytochrome c was from BD Biosciences. For Western blot analysis, radioimmune precipitation assay buffer (Sigma) was used for tissue lysis, and cell lysis buffer (Cell Signaling Technology, Inc.) was used for whole cell lysis together with protease inhibitors. Western gel images were quantified by densitometry using Image J software (National Institutes of Health). Manganese(III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP) was from Enzo Life Sciences. Caspase 1 activity was determined using a caspase 1 activity colorimetric kit (R&D Systems). Caspase 3 activity was determined using a caspase 3 activity fluorometric kit (R&D Systems)

Measurement of Autophagic Flux
Autophagic flux was assessed by increase in GFP-LC3 puncta or Western blot analysis of LC3 in hepatocytes after treatment with bafilomycin (50 nm, Sigma) for 1 h or transfecting hepatocytes with the GFP-RFP-LC3 plasmid. 24 h after transfection, cells were imaged with a Zeiss LSM 510 laser-scanning confocal microscope using a ×63 oil lens. The numbers of GFP and RFP double-positive (early autophagic vacuoles) and RFP-only (late autophagic vacuoles) puncta were counted for each cell.

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Manufacturer protocol

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