Immunofluorescence analysis of Cyclophilin D was done on 70% confluent log phase A431 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Cyclophilin D Rabbit Polyclonal Antibody (PA3023) at 1:250 dilution in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing cytoplasmic localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Human, Mouse, Rat|
|Published species reactivity||Amphibian, Mouse|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Full length, recombinant human CyP-40 expressed in E. coli.|
|Storage buffer||whole serum diluted in PBS|
|Contains||0.05% sodium azide|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|Gel Shift (GS)||Assay dependent|
|Western Blot (WB)||1:1,000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
|Western Blot (WB)||See 2 publications below|
PA3-023 detects cyclophilin 40 (CyP-40) from human, mouse and rat tissues and cells.
PA3-023 has been successfully used in Western blot and gel shift procedures. By Western blot, this antibody detects a 40 kDa protein representing CyP-40 from rat brain extract.
The PA3-023 immunogen is recombinant human CyP-40 expressed in E. coli.
Immunophilins are a family of soluble cytosolic receptors capable of binding to one of two major immunosuppressant agents: cyclosporin A (CsA) or FK506. Proteins that bind FK506 are termed FK506 Binding Proteins (FKBPs) and those that bind cyclosporin A are called cyclophilins (CyP).
Both CyP:CsA and FKBP:FK506 complexes have been shown to inhibit calcineurin, a calcium and calmodulin dependent protein phosphatase which has been implicated as an important signaling enzyme in T-cell activation, providing a possible mechanism of immunosuppression by CsA and FK506. Immunophilins function as peptidyl prolyl cis-trans-isomerases (PPIase) whose activity is inhibited by their respective immunosuppressant compounds. As PPIase's, immunophilins accelerate folding of some proteins both in vivo and in vitro by catalyzing slow steps in the initial folding and rearrangement of proline containing proteins.
CyP-40, a 40 kDa protein, shares significant homology with smaller CyPA (CyP-18) and FKBP59. CyP-40 exhibits the characteristic CsA binding and isomerase activity of CyP-18, though these activities appear to be less with CyP-40 than with Cyp-18. Like FKBP59, CyP-40 has been found in progesterone receptor complexes. CyP-40 is expressed at similar levels in many tissues.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Cyclophilin A is required for retinoic acid-induced neuronal differentiation in p19 cells.
PA3-023 was used in western blot to investigate the role of cyclophilin A in the retinoic acid-induced p19 cell differentiation.
|Song J,Lu YC,Yokoyama K,Rossi J,Chiu R||The Journal of biological chemistry (279:24414)||2004|
Multiple components of the HSP90 chaperone complex function in regulation of heat shock factor 1 In vivo.
PA3-023 was used in western blot to study the complexing process of heat shock factor 1 with the HSP90 chaperone machinery.
|Bharadwaj S,Ali A,Ovsenek N||Molecular and cellular biology (19:8033)||1999|