Revision 1
Cell Signaling Technology

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Web: [email protected] cellsignal.com

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For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
Tom20 (D8T4N) Rabbit mAb 42406 20 µl 16 kDa Rabbit IgG
OPA1 (D6U6N) Rabbit mAb 80471 20 µl 80-100 kDa Rabbit IgG
Phospho-DRP1 (Ser616) (D9A1) Rabbit mAb 4494 20 µl 78-82 kDa Rabbit IgG
DRP1 (D8H5) Rabbit mAb 5391 20 µl 78-82 kDa Rabbit IgG
Phospho-MFF (Ser146) Antibody 49281 20 µl 25, 27 kDa Rabbit 
MFF (E5W4M) XP® Rabbit mAb 84580 20 µl 25, 27, 30, 35 kDa Rabbit IgG
Mitofusin-1 (D6E2S) Rabbit mAb 14739 20 µl 82 kDa Rabbit IgG
Mitofusin-2 (D1E9) Rabbit mAb 11925 20 µl 80 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

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.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Background

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). 

  1. Chacinska, A. et al. (2009) Cell 138, 628-44.
  2. Saitoh, T. et al. (2007) EMBO J 26, 4777-87.
  3. Kasahara, A. and Scorrano, L. (2014) Trends Cell Biol 24, 761-70.
  4. Delettre, C. et al. (2001) Hum Genet 109, 584-91.
  5. Olichon, A. et al. (2007) Cell Death Differ 14, 682-92.
  6. Ishihara, N. et al. (2006) EMBO J 25, 2966-77.
  7. Cipolat, S. et al. (2006) Cell 126, 163-75.
  8. Griparic, L. et al. (2007) J Cell Biol 178, 757-64.
  9. Merkwirth, C. et al. (2008) Genes Dev 22, 476-88.
  10. Müller-Rischart, A.K. et al. (2013) Mol Cell 49, 908-21.
  11. Knott, A.B. et al. (2008) Nat Rev Neurosci 9, 505-18.
  12. Kashatus, J.A. et al. (2015) Mol Cell 57, 537-51.
  13. Kashatus, D.F. et al. (2011) Nat Cell Biol 13, 1108-15.
  14. Taguchi, N. et al. (2007) J Biol Chem 282, 11521-9.
  15. Chang, C.R. and Blackstone, C. (2007) J Biol Chem 282, 21583-7.
  16. Cribbs, J.T. and Strack, S. (2007) EMBO Rep 8, 939-44.
  17. Liu, R. and Chan, D.C. (2015) Mol Biol Cell 26, 4466-77.
  18. Shen, Q. et al. (2014) Mol Biol Cell 25, 145-59.
  19. Losón, O.C. et al. (2013) Mol Biol Cell 24, 659-67.
  20. Otera, H. et al. (2010) J Cell Biol 191, 1141-58.
  21. Toyama, E.Q. et al. (2016) Science 351, 275-281.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    XP is a registered trademark of Cell Signaling Technology, Inc.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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    Revision 1
    #48799

    Mitochondrial Dynamics Antibody Sampler Kit

    Mitochondrial Dynamics Antibody Sampler Kit: Image 1 Expand Image
    Western blot analysis of extracts from various cell lines using Mitofusin-2 (D1E9) Rabbit mAb.
    Mitochondrial Dynamics Antibody Sampler Kit: Image 2 Expand Image
    Western blot analysis of extracts from various cell lines using Mitofusin-1 (D6E2S) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Mitochondrial Dynamics Antibody Sampler Kit: Image 3 Expand Image
    Western blot analysis of extracts from HeLa and Saos-2 cells using Tom20 (D8T4N) Rabbit mAb (upper) and β-Tubulin (D2N5G) Rabbit mAb #15115 (lower).
    Mitochondrial Dynamics Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from HeLa cells, untreated (-) or treated (+) with nocodazole (100 ng/ml, 17 hr), using Phospho-DRP1 (Ser616) (D9A1) Rabbit mAb (upper) and DRP1 (D6C7) Rabbit mAb #8570 (lower).
    Mitochondrial Dynamics Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from MCF7 cells, untreated (-) or treated with CCCP (100μM, 2hr; +), using phospho-MFF (Ser146) Antibody (upper) or total MFF antibody (lower).
    Mitochondrial Dynamics Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from various cell lines using DRP1 (D8H5) Rabbit mAb.
    Mitochondrial Dynamics Antibody Sampler Kit: Image 7 Expand Image
    After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
    Mitochondrial Dynamics Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis of extracts of MEFs from wild-type (-) and OPA knockout (OPA-/-; +) mice using OPA1 (D6U6N) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Mitochondrial Dynamics Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with siRNA targeting human MFF (+), using MFF (E5W4M) XP® Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
    Mitochondrial Dynamics Antibody Sampler Kit: Image 10 Expand Image
    Immunoprecipitation of Mitofusin-2 from PANC-1 cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Mitofusin-2 (D1E9) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Mitofusin-2 (D2D10) Rabbit mAb #9482.
    Mitochondrial Dynamics Antibody Sampler Kit: Image 11 Expand Image
    Western blot analysis of extracts from HeLa cells, transfected with SignalSilence® Control siRNA (unconjugated) #6568 (-), or SignalSilence® Mitofusin-1 siRNA I #13274 (+), using Mitofusin-1 (D6E2S) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). The Mitofusin-1 (D6E2S) Rabbit mAb confirms silencing of mitofusin-1 expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.
    Mitochondrial Dynamics Antibody Sampler Kit: Image 12 Expand Image
    Immunohistochemical analysis of paraffin-embedded HeLa cell pellet (left, high-expressing) or Saos-2 cell pellet (right, low-expressing) using Tom20 (D8T4N) Rabbit mAb.
    Mitochondrial Dynamics Antibody Sampler Kit: Image 13 Expand Image
    Confocal immunofluorescent analysis of HeLa cells, untreated (left) or λ phosphatase-treated (right), using Phospho-DRP1 (Ser616) (D9A1) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Mitochondrial Dynamics Antibody Sampler Kit: Image 14 Expand Image
    Western blot analysis of extracts from MCF7 cells, untreated (-) or treated with CCCP (100uM, 2hr; +), using Phospho-MFF (Ser146) Antibody. The phospho-specificity of the antibody was verified by treating the membrane with (+) or without (-) calf intestinal phosphatase (CIP) after protein transfer.
    Mitochondrial Dynamics Antibody Sampler Kit: Image 15 Expand Image
    Immunoprecipitation of DRP1 from NIH/3T3 cell extracts using DRP1 (D8H5) Rabbit mAb (lane 2). Western blot detection was performed using the same antibody. Lane 1 is 10% input.
    Mitochondrial Dynamics Antibody Sampler Kit: Image 16 Expand Image
    Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® OPA1 siRNA I (+), or SignalSilence® OPA1 siRNA II using OPA1 (D6U6N) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The OPA1 (D6U6N) Rabbit mAb confirms silencing of OPA1 expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.
    Mitochondrial Dynamics Antibody Sampler Kit: Image 17 Expand Image
    Western blot analysis of extracts from wild-type mouse embryonic fibroblasts (MEFs) (WT), Mff (-/-) MEFs (Mff KO), or Mff (-/-) MEFs reconstituted with a construct expressing human MFF protein, isoform 5 (Mff KO+hMFF) using MFF (E5W4M) XP® Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). Lysates courtesy of Sebastien Herzig and Portia Lombardo, Reuben Shaw lab, Salk Institute.
    Mitochondrial Dynamics Antibody Sampler Kit: Image 18 Expand Image
    Confocal immunofluorescent analysis of HeLa cells using Mitofusin-2 (D1E9) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Mitochondrial Dynamics Antibody Sampler Kit: Image 19 Expand Image
    Immunoprecipitation of mitofusin-1 protein from HeLa cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Mitofusin-1 (D6E2S) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Mitofusin-1 (D6E2S) Rabbit mAb.
    Mitochondrial Dynamics Antibody Sampler Kit: Image 20 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse kidney using Tom20 (D8T4N) Rabbit mAb.
    Mitochondrial Dynamics Antibody Sampler Kit: Image 21 Expand Image
    Flow cytometric analysis of Jurkat cells, untreated (left) or λ phosphatase-treated (right), using Phospho-DRP1 (Ser616) (D9A1) Rabbit mAb and propidium iodide (DNA content).
    Mitochondrial Dynamics Antibody Sampler Kit: Image 22 Expand Image
    Western blot analysis of extracts from MCF7 and HeLa cells, untreated (-) or treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP; +) using OPA1 (D6U6N) Rabbit mAb.
    Mitochondrial Dynamics Antibody Sampler Kit: Image 23 Expand Image
    Western blot analysis of extracts from various cell lines using MFF (E5W4M) XP® Rabbit mAb.
    Mitochondrial Dynamics Antibody Sampler Kit: Image 24 Expand Image
    Confocal immunofluorescent analysis of HeLa cells using Mitofusin-1 (D6E2S) Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Mitochondrial Dynamics Antibody Sampler Kit: Image 25 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung adenocarcinoma using Tom20 (D8T4N) Rabbit mAb.
    Mitochondrial Dynamics Antibody Sampler Kit: Image 26 Expand Image
    Western blot analysis of extracts from various cell lines using OPA1 (D6U6N) Rabbit mAb.
    Mitochondrial Dynamics Antibody Sampler Kit: Image 27 Expand Image
    Immunoprecipitation of MFF from HeLa cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is MFF (E5W4M) XP® Rabbit mAb. Western blot analysis was performed using MFF (E5W4M) XP® Rabbit mAb. Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used for detection to avoid cross-reactivity with IgG.
    Mitochondrial Dynamics Antibody Sampler Kit: Image 28 Expand Image
    Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using Tom20 (D8T4N) Rabbit mAb.
    Mitochondrial Dynamics Antibody Sampler Kit: Image 29 Expand Image
    Immunoprecipitation of OPA1 from MCF7 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is OPA1 (D6U6N) Rabbit mAb. Western blot analysis was performed using OPA1 (D6U6N) Rabbit mAb. A conformation specific secondary antibody was used to avoid reactivity with IgG.
    Mitochondrial Dynamics Antibody Sampler Kit: Image 30 Expand Image
    Confocal immunofluorescent analysis of wild-type mouse embryonic fibroblasts (MEFs) (WT, left), Mff (-/-) MEFs (Mff KO, center), or Mff (-/-) MEFs reconstituted with a construct expressing human MFF protein, isoform 5 (Mff KO+hMFF, right) using MFF (E5W4M) XP® Rabbit mAb (green). Nuclei labeled with DAPI (blue). Image courtesy of Sebastien Herzig and Portia Lombardo, Reuben Shaw lab, Salk Institute.
    Mitochondrial Dynamics Antibody Sampler Kit: Image 31 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse small intestine using Tom20 (D8T4N) Rabbit mAb.
    Mitochondrial Dynamics Antibody Sampler Kit: Image 32 Expand Image
    Confocal immunofluorescent analysis of HeLa cells using MFF (E5W4M) XP® Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Mitochondrial Dynamics Antibody Sampler Kit: Image 33 Expand Image
    Confocal immunofluorescent analysis of HeLa (high expression, left) and Saos-2 (low expression, right) cells, using Tom20 (D8T4N) Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).