Western blot analysis of LC3 Control Cell Extracts from HeLa cells, untreated (-) or chloroquine-treated (50 μM, overnight; +), using LC3B (D11) XP® Rabbit mAb #3868 (upper), LC3A/B Antibody #4108 (middle), or β-Tubulin (9F3) Rabbit mAb #2128 (lower).
Boil for 3 minutes prior to use. Load 10 µl of untreated and chloroquine treated LC3 Control Cell Extracts per lane.
Supplied in SDS Sample Buffer: 62.5 mM Tris- HCl (pH 6.8 at 25°C), 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue or phenol red.Store at –20°C, or at –80°C for long-term storage.
LC3 Control Cell Extracts (HeLa Untreated): Total cell extracts from HeLa cells serve as a negative control. Supplied in SDS sample buffer.
LC3 Control Cell Extracts (HeLa +Chloroquine): Total cell extracts from HeLa cells treated with 50 μM chloroquine overnight serve as a positive control.
This lysate pair is produced as a control for western blotting of LC3A and LC3B. LC3C cannot be detected in these lysates.
Autophagy is a catabolic process for the autophagosomic-lysosomal degradation of bulk cytoplasmic contents (1,2). Autophagy is generally activated by conditions of nutrient deprivation, but it has also been associated with a number of physiological processes including development, differentiation, neurodegenerative diseases, infection, and cancer (3). Autophagy marker Light Chain 3 (LC3) was originally identified as a subunit of microtubule-associated proteins 1A and 1B (termed MAP1LC3) (4) and subsequently found to contain similarity to the yeast protein Apg8/Aut7/Cvt5 critical for autophagy (5). Three human LC3 isoforms (LC3A, LC3B, and LC3C) undergo post-translational modifications during autophagy (6-9). Cleavage of LC3 at the carboxy terminus immediately following synthesis yields the cytosolic LC3-I form. During autophagy, LC3-I is converted to LC3-II through lipidation by a ubiquitin-like system involving Atg7 and Atg3 that allows for LC3 to become associated with autophagic vesicles (6-10). The presence of LC3 in autophagosomes and the conversion of LC3 to the lower migrating form, LC3-II, have been used as indicators of autophagy (11).
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