Each antibody in the Mitophagy Antibody Sampler kit detects endogenous levels of its target protein. LC3B (D11) XP® Rabbit mAb detects type I and type II forms of LC3B. Weaker reactivity is observed with rodent LC3B.
Source / Purification
Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Gly162 of human SQSTM1/p62, Val135 of human NDP52, Pro140 of human PINK1, Leu410 of human Optineurin, Glu128 of human BNIP3L/Nix, peptide sequences corresponding to residues near the amino terminus of human LC3B, the amino terminus of human BNIP3, a recombinant fusion protein with an epitope that maps to the carboxy terminus of human Parkin, and a synthetic phospho-peptide corresponding to residues surrounding Ser65 of human Ubiquitin protein. Antibodies are purified by protein A and peptide affinity chromatography.
Autophagy is a catabolic process for the autophagosome-lysosomal degradation of bulk cytoplasmic contents (1, 2). Selective autophagy targets the degradation of distinct sets of substrates and organelles (3-5). One of the best studied examples of selective autophagy involves the clearance of damaged mitochondria through a process called mitophagy. Several pathways have been described for various contexts of mitophagy, including the FUNDC1 pathway, the BNIP3 and BNIP3L/Nix pathway, and the PINK1/Parkin pathway. FUNDC1 is a mitochondrial protein that is phosphorylated by the autophagy kinase ULK1 and regulates hypoxia induced mitophagy (6, 7). BNIP3L/Nix and BNIP3 are members of the Bcl-2 family of apoptosis regulators that are expressed on mitochondria, induced by hypoxia, and have been shown to play a role in mitophagy (8). BNIP3L/Nix is also important in the autophagic maturation of erythroid cells (9). FUNDC1, BNIP3 and BNIP3L/Nix bind to LC3 family members, targeting the mitochondria to the autophagosome.Non-hypoxic induction of mitophagy can be regulated by the PINK1/Parkin pathway, which plays causative roles in neurodegenerative disease, most notably Parkinson’s disease (10, 11). PINK1 is a mitochondrial serine/threonine kinase that is stabilized on the outer mitochondrial membrane of damaged mitochondria. Substrates of PINK1 include the E3 ubiquitin ligase Parkin and ubiquitin itself (12-14). Phosphorylation of Parkin as well as binding to phosphorylated ubiquitin leads to accumulation of ubiquitinated chains on multiple mitochondrial proteins. Ubiquitinated proteins are recognized by selective cargo receptors including SQSTM1/p62, Optineurin, and NDP52 (15-16). Autophagy cargo receptors contain an LC3-interacting region (LIR) required for binding to Atg8/LC3 family members and targeting to the autophagosome (3). The Mitophagy Antibody Sampler Kit provides an economical means of detecting proteins involved in the process of mitophagy. The kit includes enough antibody to perform two western blot experiments with each primary antibody.