Purification notesPurified from rabbit antiserum by affinity chromatography using epitope specific phosphopeptide. The antibody against non-phosphopeptide was removed by chromatography using non-phosphopeptide corresponding to the phosphorylation site.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/500 - 1/1000. Detects a band of approximately 55 kDa (predicted molecular weight: 55 kDa).
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
FunctionEssential component of an SCF-type E3 ligase complex, SCF(NIPA), a complex that controls mitotic entry by mediating ubiquitination and subsequent degradation of cyclin B1 (CCNB1). Its cell-cycle-dependent phosphorylation regulates the assembly of the SCF(NIPA) complex, restricting CCNB1 ubiquitination activity to interphase. Its inactivation results in nuclear accumulation of CCNB1 in interphase and premature mitotic entry. May have an antiapoptotic role in NPM-ALK-mediated signaling events.
Tissue specificityWidely expressed. Highly expressed in heart, skeletal muscle and testis. Expressed in brain, placenta, lung, kidney, liver, pancreas, spleen, thymus, prostate, ovary small intestine and colon. Weakly or not expressed in leukocytes.
PathwayProtein modification; protein ubiquitination.
Developmental stageWeakly expressed in G0/G1 phases, abundant during S and G2/M phases, and strongly decreases thereafter.
DomainThe F-box-like region is required for the interaction with SKP1.
Post-translational modificationsPhosphorylated. Phosphorylated on Ser residues at G2/M phase, but not during S and G0 phases. May also be weakly phosphorylated on Tyr residues. Ser-354 phosphorylation, a major site during the course of cell-cycle-dedendent phosphorylation, results in its dissociation from the SCF(NIPA) complex, thereby preventing CCNB1 degradation leading to mitotic entry.
IHC image of NIPA staining in human testis formalin fixed paraffin embedded tissue section, performed on a Leica Bond system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab63557, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
Western blot - NIPA (phospho S354) antibody (ab63557)
All lanes : Anti-NIPA (phospho S354) antibody (ab63557) at 1/500 dilution
Lane 1 : extracts from COS7 cells, treated
with HU (2nM, 24hours) Lane 2 : extracts from COS7 cells, treated
with HU (2nM, 24hours) with immunizing phosphopeptide at 10 µg
Lysates/proteins at 30 µg per lane.
Predicted band size : 55 kDa Observed band size : 55 kDa Additional bands at : 30 kDa,75 kDa. We are unsure as to the identity of these extra bands.
References for Anti-NIPA (phospho S354) antibody (ab63557)
This product has been referenced in:
Zhao M et al. Methylene blue exerts a neuroprotective effect against traumatic brain injury by promoting autophagy and inhibiting microglial activation. Mol Med Rep13:13-20 (2016).
Read more (PubMed: 26572258) »
Illert AL et al. Extracellular signal-regulated kinase 2 (ERK2) mediates phosphorylation and inactivation of nuclear interaction partner of anaplastic lymphoma kinase (NIPA) at G2/M. J Biol Chem287:37997-8005 (2012).
Read more (PubMed: 22955283) »