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48799
Mitochondrial Dynamics Antibody Sampler Kit

Mitochondrial Dynamics Antibody Sampler Kit #48799

Western Blotting Image 1

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).

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Western Blotting Image 2

Western blot analysis of extracts from MCF7 and HeLa cells, untreated (-) or treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP; +) using OPA1 (D6U6N) Rabbit mAb.

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Western Blotting Image 3

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).

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Western Blotting Image 4

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).

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Western Blotting Image 5

Western blot analysis of extracts from various cell lines using DRP1 (D6C7) Rabbit mAb.

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Western Blotting Image 6

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).

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Western Blotting Image 7

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).

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Western Blotting Image 8

Western blot analysis of extracts from various cell lines using Mitofusin-1 (D6E2S) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).

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IF-IC Image 9

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).

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Western Blotting Image 10

Western blot analysis of extracts from various cell lines using Mitofusin-2 (D1E9) Rabbit mAb.

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Western Blotting Image 11

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.

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IHC-P (paraffin) Image 12

Immunohistochemical analysis of paraffin-embedded HeLa cell pellet (left, high-expressing) or Saos-2 cell pellet (right, low-expressing) using Tom20 (D8T4N) Rabbit mAb.

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Western Blotting Image 13

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.

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Flow Cytometry Image 14

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).

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IP Image 15

Immunoprecipitation of extracts from HCT 116 cells using DRP1 (D6C7) Rabbit mAb. The western blot was probed using the same antibody. Lane 1 is 10% input.

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Western Blotting Image 16

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.

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Western Blotting Image 17

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.

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Western Blotting Image 18

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.

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IP Image 19

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.

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IF-IC Image 20

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).

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IF-IC Image 21

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).

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IF-IC Image 22

Confocal immunofluorescent analysis of ACHN cells using DRP1 (D6C7) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Western Blotting Image 23

Western blot analysis of extracts from various cell lines using MFF (E5W4M) XP® Rabbit mAb.

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IP Image 24

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.

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Western Blotting Image 25

Western blot analysis of extracts from various cell lines using OPA1 (D6U6N) Rabbit mAb.

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IP Image 26

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.

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IF-IC Image 27

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).

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IP Image 28

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.

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IF-IC Image 29

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.

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IF-IC Image 30

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).

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Tom20 (D8T4N) Rabbit mAb 42406 20 µl
  • WB
  • IP
  • IHC
  • IF
H M R Mk 16 Rabbit IgG
OPA1 (D6U6N) Rabbit mAb 80471 20 µl
  • WB
  • IP
H M R 80-100 Rabbit IgG
Phospho-DRP1 (Ser616) (D9A1) Rabbit mAb 4494 20 µl
  • WB
  • IF
  • F
H 78-82 Rabbit IgG
DRP1 (D6C7) Rabbit mAb 8570 20 µl
  • WB
  • IP
  • IF
H M R Mk 78-82 Rabbit IgG
Phospho-MFF (Ser146) Antibody 49281 20 µl
  • WB
H M R 25, 27 Rabbit 
MFF (E5W4M) XP® Rabbit mAb 84580 20 µl
  • WB
  • IP
  • IF
H M R 25, 27, 30, 35 Rabbit IgG
Mitofusin-1 (D6E2S) Rabbit mAb 14739 20 µl
  • WB
  • IP
  • IF
H 82 Rabbit IgG
Mitofusin-2 (D1E9) Rabbit mAb 11925 20 µl
  • WB
  • IP
  • IF
H Hm Mk 80 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

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 and DRP1, surrounding Leu821 of human OPA1, Pro126 of human MFF isoform 1, Pro551 of human mitofusin-1, and Val573 of human mitofusin-2, and a synthetic phosphopeptide surrounding Ser616 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). 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) (15-18). AMPK directly phosphorylates MFF at two sites to allow for enhanced recruitment of Drp1 to the mitochondria (19). 

  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. Liu, R. and Chan, D.C. (2015) Mol Biol Cell 26, 4466-77.
  16. Shen, Q. et al. (2014) Mol Biol Cell 25, 145-59.
  17. Losón, O.C. et al. (2013) Mol Biol Cell 24, 659-67.
  18. Otera, H. et al. (2010) J Cell Biol 191, 1141-58.
  19. Toyama, E.Q. et al. (2016) Science 351, 275-281.
Entrez-Gene Id
10059 , 56947 , 55669 , 9927 , 4976 , 9804
Swiss-Prot Acc.
O00429 , Q9GZY8 , Q9GZY8-2 , Q9GZY8-5 , Q8IWA4 , O95140 , O60313 , Q15388
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.

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