Cell Signaling Technology

Product Pathways - Vesicle Trafficking

Vesicle Trafficking Antibody Sampler Kit #9765

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
Phospho-Caveolin-1 (Tyr14) Antibody #3251 40 µl W H M R Mk (Dg) 23, 25 Rabbit
Caveolin-1 (D46G3) XP® Rabbit mAb #3267 40 µl W IP IHC-P IF-IC F H M R Hm Mk B Dg 21, 24 Rabbit IgG
Clathrin Heavy Chain (D3C6) XP® Rabbit mAb #4796 40 µl W IP IF-IC H M R Mk (Pg) 190 Rabbit IgG
APPL1 (D83H4) XP® Rabbit mAb #3858 40 µl W IP IF-IC H M R Mk 82 Rabbit IgG
EEA1 (C45B10) Rabbit mAb #3288 40 µl W IP IF-IC H M R 170 Rabbit IgG
Syntaxin 6 (C34B2) Rabbit mAb #2869 40 µl W IP IF-IC H M R 32 Rabbit IgG
GOPC (D10A12) Rabbit mAb #8576 40 µl W H 59 Rabbit IgG
Rab5 (C8B1) Rabbit mAb #3547 40 µl W IF-IC H M R Mk 25 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Hm=Hamster  Mk=Monkey  B=Bovine  Dg=Dog  Pg=Pig
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

Phospho-Caveolin-1 (Tyr14) Antibody detects endogenous levels of Caveolin-1 only when phosphorylated at Tyr14 and does not cross-react with caveolin-2, -3 or caveolin-1β, the short isoform of caveolin-1. Caveolin-1 (D46G3) XP® Rabbit mAb detects endogenous levels of total caveolin-1 protein. Clathrin Heavy Chain (D3C6) XP® Rabbit mAb detects endogenous levels of total clathrin protein. APPL1 (D83H4) XP® Rabbit mAb detects endogenous levels of total APPL1 protein. EEA1 (C45B10) Rabbit mAb detects endogenous levels of total EEA1 protein. Syntaxin 6 (C34B2) Rabbit mAb detects endogenous levels of total Syntaxin 6 protein. GOPC (D10A12) Rabbit mAb detects endogenous levels of total GOPC protein. Rab5 (C8B1) Rabbit mAb detects endogenous levels of total Rab5 protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Syntaxin 6 (C34B2) Rabbit mAb #2869.

Western Blotting

Western Blotting

Western blotting analysis of extracts from HeLa cells treated with H2O2 for the indicated times using Phospho-Caveolin-1 (Tyr14) Antibody #3251 (upper) or caveolin-1 antibody (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell types using Caveolin-1 (D46G3) XP® Rabbit mAb #3267.


Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using EEA1 (C45B10) Rabbit mAb #3288.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Rab5 (C8B1) Rabbit mAb #3547.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell types using APPL1 (D83H4) XP® Rabbit mAb #3858.


Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Clathrin Heavy Chain (D3C6) XP® Rabbit mAb #4796.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using GOPC (D10A12) Rabbit mAb #8576.

Description

The Vesicle Trafficking Antibody Sampler kit provides an economical means to analyze proteins involved in the intracellular transport of cargo proteins. This kit includes enough primary and secondary antibody to perform four western blot experiments.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Tyr14 of human caveolin-1. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Glu20 of human caveolin-1, Pro1663 of human clathrin heavy chain protein, Thr426 of human APPL1, Ser70 of human EEA1 protein, Tyr140 of mouse syntaxin 6 protein, Phe31 of human GOPC protein, and Gly190 of human Rab5 protein.

Background

Vesicle trafficking is an integral cellular process and the associated proteins involved also play major roles in other signaling pathways. Caveolins are involved in diverse biological functions including vesicular trafficking, cholesterol homeostasis, cell adhesion, apoptosis, and are also indicated in neurodegenerative disease (1). It is believed that caveolins serve as scaffolding proteins for the integration of signal transduction. Phosphorylation at Tyr14 is essential for caveolin association with SH2 or PTB domain-containing adaptor proteins, such as GRB7 (2-4).Clathrin-coated vesicles provide for the intracellular transport of proteins following endocytosis and during multiple vesicle trafficking pathways. Vesicles form at specialized areas of the cell membrane where clathrin and associated proteins form clathrin-coated pits. Invagination of these cell membrane-associated pits internalizes proteins and forms an intracellular clathrin-coated vesicle (5,6). Clathrin is the most abundant protein in these vesicles and is present as a basic assembly unit called a triskelion. Each clathrin triskelion is composed of three clathrin heavy chains and three clathrin light chains. Clathrin heavy chain proteins are composed of several functional domains that associate with other vesicle proteins (6).The APPL1 multidomain adaptor protein is a BAR-domain protein family member that is involved in membrane trafficking within a number of signal transduction pathways (7).EEA1 is an early endosomal marker and a Rab5 effector protein essential for early endosomal membrane fusion and trafficking (8,9). Syntaxin 6 is a ubiquitously expressed S25C family member of the SNARE proteins (10,11). Syntaxin 6 protein is localized to the trans-Golgi and within endosomes and regulates membrane trafficking by partnering with a variety of other SNARE proteins (12-14). It has two coiled-coil domains (CC1 and CC2) located in the amino-terminal region and a PDZ domain in the carboxy-terminal region (15). The CC2 domain and its adjacent linker region mediate the association of GOPC with the Golgi protein golgin-160 and the Q-SNARE protein syntaxin 6 (15,16). The PDZ domain of GOPC interacts with the carboxy terminus of target proteins to mediate target protein vesicular trafficking and surface expression (17-20).Rab5 is a member of the Ras superfamily of small Rab GTPases. Rab5 is localized at the plasma membrane and early endosomes and functions as a key regulator of vesicular trafficking during early endocytosis (21).

  1. Smart, E.J. et al. (1999) Mol Cell Biol 19, 7289-304.
  2. Nomura, R. and Fujimoto, T. (1999) Mol Biol Cell 10, 975-86.
  3. Volonté, D. et al. (2001) J Biol Chem 276, 8094-103.
  4. Lee, H. et al. (2000) Mol Endocrinol 14, 1750-75.
  5. Rodriguez-Boulan, E. et al. (2005) Nat Rev Mol Cell Biol 6, 233-47.
  6. Mousavi, S.A. et al. (2004) Biochem J 377, 1-16.
  7. Habermann, B. (2004) EMBO Rep 5, 250-5.
  8. Mu, F.T. et al. (1995) J Biol Chem 270, 13503-11.
  9. Christoforidis, S. et al. (1999) Nature 397, 621-5.
  10. Bock, J.B. et al. (2001) Nature 409, 839-41.
  11. Bock, J.B. et al. (1996) J Biol Chem 271, 17961-5.
  12. Wendler, F. and Tooze, S. (2001) Traffic 2, 606-11.
  13. Bock, J.B. et al. (1997) Mol Biol Cell 8, 1261-71.
  14. Mallard, F. et al. (2002) J Cell Biol 156, 653-64.
  15. Charest, A. et al. (2001) J Biol Chem 276, 29456-65.
  16. Hicks, S.W. and Machamer, C.E. (2005) J Biol Chem 280, 28944-51.
  17. Cheng, J. et al. (2002) J Biol Chem 277, 3520-9.
  18. He, J. et al. (2004) J Biol Chem 279, 50190-6.
  19. Wente, W. et al. (2005) J Biol Chem 280, 32419-25.
  20. Ito, H. et al. (2006) Biochem J 397, 389-98.
  21. Zerial, M. and McBride, H. (2001) Nat Rev Mol Cell Biol 2, 107-17.

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