USP9x can be detected in the polarized intestinal cell line T84 which is a human colon carcinoma epithelial cell line. In tissue culture: USP9x is also observed in L-cell fibroblasts and MDCK cells. USP9x is ubiquitously expressed in all adult tissues.
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration.
Use a concentration of 1 µg/ml. Detects a band of approximately 100,105, 290 kDa (predicted molecular weight: 54, 100,105 , 290 kDa).
IHC (PFA fixed)
Use a concentration of 0.1 µg/ml.
Use a concentration of 5 µg/ml.
Use at an assay dependent concentration.
Deubiquitinase involved both in the processing of ubiquitin precursors and of ubiquitinated proteins. May therefore play an important role regulatory role at the level of protein turnover by preventing degradation of proteins through the removal of conjugated ubiquitin. Essential component of TGF-beta/BMP signaling cascade. Regulates chromosome alignment and segregation in mitosis by regulating the localization of BIRC5/survivin to mitotic centromeres. Specifically hydrolyzes both 'Lys-29'- and 'Lys-33'-linked polyubiquitins chains. Specifically deubiquitinates monoubiquitinated SMAD4, opposing the activity of E3 ubiquitin-protein ligase TRIM33.
Uubiquitin specific protease 9, X chromosome (fat facets like Drosophila) antibody
X chromosome antibody
Western blot - Anti-USP9x antibody (ab19879)
Lane 1: Wild-type HAP1 cell lysate (20 µg) Lane 2: USP9x knockout HAP1 cell lysate (20 µg) Lane 3: T84 cell lysate (20 µg) Lane 4: NIH3T3 cell lysate (20 µg) Lanes 1 to 4: Merged signal (red and green). Green - ab19879 observed at 290 kDa. Red - loading control, ab181602 observed at 124 kDa. ab19879 was shown to specifically react with USP9x when USP9x knockout samples were used. Wild-type and USP9x knockout samples were subjected to SDS-PAGE. ab9879 and ab181602 (loading control to GAPDH) were both diluted at 1 µg/ml and 1/10000 respectively and incubated overnight at 4°C. Blots were developed withGoat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.
ab19879 detects full length USP9x protein as well as a number of USP9x fragments in WB on Caco2 Lysate:
289kDa Human protein: Q93008 USP9X (Full length protein) 105kDa Human protein: Q6P468 - USP9X protein (Fragment) Human protein 99.7kDa Human protein: Q59EZ5 - USP9X protein variant (Fragment). 53.9kDa Q86X58 - USP9X protein (Fragment).
Immunohistochemistry (PFA fixed) - Anti-USP9x antibody (ab19879)This image is courtesy of Sophie Pezet, King's College London, United Kingdom
Immuofluorescent staining for USP9X in the rat hippocampus (dentate gyrus) using ab19879 (1/300 = 0.07µg/ml). Image is taken with X10 objective. ab19879 was incubated overnight at RT. Secondary antibody used was anti-rabbit Alexa fluor 488 (1/1000 for 2h at RT). Rats were intracardially perfused with paraformaldehyde 4%, brain tissue was post-fixed overnight in the same fixative, cryoprotected in 20% sucrose and frozen in OCT. 30µm coronal sections were cut on a cryostat for free floating IHC.
ICC/IF image of ab19879 stained Hek293 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab19879, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
USP9x was immunoprecipitated using 0.5mg Caco2 whole cell extract, 5µg of Rabbit polyclonal to USP9x and 50µl of protein G magnetic beads (+). No antibody was added to the control (-). The antibody was incubated under agitation with Protein G beads for 10min, Caco2 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation. Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab19879. Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697). Band: 290kDa: USP9x.
Reijnders MR et al. De Novo Loss-of-Function Mutations in USP9X Cause a Female-Specific Recognizable Syndrome with Developmental Delay and Congenital Malformations. Am J Hum Genet98:373-81 (2016).
Read more (PubMed: 26833328) »
Yang B et al. Deubiquitinase USP9X deubiquitinates ß-catenin and promotes high grade glioma cell growth. Oncotarget7:79515-79525 (2016).
Read more (PubMed: 27783990) »