Immunofluorescence analysis of PSMD2 was performed using 70% confluent log phase Hep G2 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 PSMD2 Rabbit Polyclonal Antibody (PA1962) 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 (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 represents the merged image showing cytoplasmic localization. Panel e shows the control without primary antibody. The images were captured at 60X magnification.
|Tested species reactivity||Dog, Hamster, Human, Mouse, Non-human primate, Rat|
|Published species reactivity||Not Applicable|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic Peptide: C A(278) T N R P D T L D P A L L R P G R(294)|
|Purification||Ammonium sulfate precipitation|
|Contains||0.05% sodium azide|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|Western Blot (WB)||1:500-1:2,000|
* 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.
|Miscellaneous PubMed (MISC)||See 1 publications below|
PA1-962 detects proteasome 19S subunit 10B from canine, hamster, human, mouse and rat tissues and cells.
PA1-962 has been successfully used in Western blot procedures. By Western blot, this antibody detects ~42, 46, 48, and 52 kDa proteins which correspond to subunit 10B and probably the combined signal from 4, 6B, 7, and 8 subunit proteins.
PA1-962 immunizing peptide corresponds to amino acid residues 278-294 of human proteasome 19S subunit 10B. This sequence is conserved in 19S protein subunits S4 (~53 kDa), S6A (~53 kDa), S6B (~46 kDa), S7 (~47 kDa), and S8 (~45 kDa). PA1-962 immunizing peptide (Cat. # PEP-107) is available for use in neutralization and control experiments.
Proteolytic degradation is critical to the maintenance of appropriate levels of short-lived and regulatory proteins as important and diverse as those involved in cellular metabolism, heat shock and stress response, antigen presentation, modulation of cell surface receptors and ion channels, cell cycle regulation, transcription, and signalling factors. The ubiquitin-proteasome pathway deconstructs most proteins in the eukaryotic cell cytosol and nucleus. Others are degraded via the vacuolar pathway which includes endosomes, lysosomes, and the endoplasmic reticulum.
The 26S proteasome is an ATP-dependent, multisubunit (~31), barrel-shaped molecular machine with an apparent molecular weight of ~2.5 MDa. It consists of a 20S proteolytic core complex which is crowned at one or both ends by 19S regulatory subunit complexes. The 19S regulatory subunits recognize ubiquitinated proteins and play an essential role in unfolding and translocating targets into the lumen of the 20S subunit. An enzymatic cascade is responsible for the attachment of multiple ubiquitin molecules to lysine residues of proteins targeted for degradation. Several genetic diseases are associated with defects in the ubiquitin-proteasome pathway. Some examples of affected proteins include those linked to cystic fibrosis, Angelman and quote;s syndrome, and Liddle syndrome.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
|Not Applicable||Not Cited||
The cyclophilin-like domain of Ran-binding protein-2 modulates selectively the activity of the ubiquitin-proteasome system and protein biogenesis.
PA1-962 was used in western blot to investigate the role of proteasome subunits S10B during the proteolysis of intracellular proteins regulated by the ubiquitin-proteasome system.
|Yi H,Friedman JL,Ferreira PA||The Journal of biological chemistry (282:34770)||2007|