|74792||Mitochondrial Dynamics Antibody Sampler Kit II||
The Mitochondrial Dynamics Antibody Sampler Kit provides an economical means to examine signaling involved in mitochondrial dynamics. The kit contains enough primary antibody to perform two western blot experiments.
Each antibody in the Mitochondrial Dynamics Antibody Sampler Kit recognizes endogenous levels of its specific target protein. Phospho-MFF (Ser146) Antibody recognizes human MFF isoforms 2, 4, and 5 but not phosphorylated Ser172 of human MFF isoform 1. This antibody cross-reacts with a 140 kDa protein of unknown identity. Based upon sequence alignment, MFF (E5W4M) XP® Rabbit mAb is predicted to react with isoforms 1-5 of human MFF protein and isoforms 1-4 of mouse MFF protein.
Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of Tom20, surrounding Leu821 of human OPA1, Pro126 of human MFF isoform 1, Pro551 of human mitofusin-1, and Val573 of human mitofusin-2, a synthetic phosphopeptide surrounding Ser616 of human DRP1, and a recombinant protein specific to the carboxy terminus of human DRP1. Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser146 of human MFF isoform 2 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Import of proteins into the mitochondria is regulated by the translocase of the outer mitochondrial membrane (TOM) complex, which facilitates transport through the outer mitochondrial membrane, and a complementary translocase of the inner membrane (TIM) complex, responsible for protein transport to the mitochondrial matrix. The TOM complex consists of the receptors Tom20, Tom22, and Tom70, and the channel-forming protein Tom40 (1). Tom20 is localized in the outer mitochondrial membrane and initially recognizes precursors with a presequence to facilitate protein import across the outer mitochondrial membrane (2).
Changes in mitochondrial dynamics regulated by environmental cues affect mitochondrial size and shape and have been shown to dramatically impact mitochondrial metabolism, apoptosis, and autophagy (3). These processes are largely controlled by mitochondrial dynamin-related GTPases, including mitofusin-1, mitofusin-2, OPA1, and DRP1. DRP1 regulates mitochondrial fission, while the mitofusins and OPA1 control fusion at the outer and inner mitochondrial membrane, respectively. These proteins are tightly regulated. OPA1 activity is regulated through alternative splicing and post-translational modifications, including complex proteolytic processing by multiple proteases (4-9). In addition, OPA1 expression can be induced under conditions of metabolic demand through a pathway involving Parkin induced NF-κB activation (10). DRP1 is regulated in part through multiple phosphorylation sites (11). Phosphorylation of DRP1 at Ser616 by MAPK or during mitosis by CDKs stimulates mitochondrial fission (12-14). In contrast, PKA dependent phosphorylation of DRP1 at Ser637 inhibits its GTPase activity and mitochondrial fission (15,16). Mitochondrial fission factor (MFF) is a tail-anchored protein that resides within the outer mitochondrial membrane and is part of the mitochondrial fission complex. MFF participates in mitochondrial fission by serving as one of multiple receptors for the GTPase dynamin-related protein 1 (Drp1) (17-20). AMPK directly phosphorylates MFF at two sites to allow for enhanced recruitment of Drp1 to the mitochondria (21).
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