Phospho Sling shot 1

Slingshot 1 is a member of slingshot (SSH) family of protein phosphatases; it is located at human chromosome 12q24.11. Slingshot is one of the primary dedicated cofilin phosphatases. It plays an important role in cell migration by dephosphorylating cofilin at Ser3 and activating it in a variety of cell types such as keratinocytes and aortic smooth muscle cells (1). Slingshot (SSH) activity can be modulated by F-actin binding, calcium acting via calcinerium, and by its association in inhibitory complexes with 14-3-3 regulatory proteins (2). 14-3-3zeta/tau heterodimers are key regulators of slingshot 1 isoform 1 (SSH1) activity in keratinocytes (3). SSH1 is involved in H₂O₂ induced cofilin dephosphorylation and activation. Such activation of SSH1-cofilin pathway stimulates the SSH1 dependent formation of cofilin-actin rods in HeLa cells (4). By dephosphorylating and activating cofilin, SSH1 plays a critical role in insulin-induced membrane protrusion and cytokinesis.

SSH1 has the SSH family-specific, N-terminal, noncatalytic (SSH-N) domain. This domain has been shown to play critical roles in P-cofilin recognition, F-actin-mediated activation, and subcellular localization of SSH1 (5). Recently it was shown that expression of phosphatase-dead versions of SSH proteins resulted in phosphorylation/inactivation of cofilin, changes in cytoskeleton organization, loss of cell polarity, and assembly of aberrant arrays of laminin-332 in human keratinocytes (6). SSH1 mutations were seen in patients with disseminated superficial actinic porokeratosis. SSH1 is expressed in a various cell types including keratinocytes, HeLa cells and smooth muscle cells. It is approximately a 126kDa protein (1049 amino acids).  The Slingshot 1 portein is phosphorylated at Serine    and phosphorylation cuases activation in the phosphatse activity. 

The Phospho-Slinshot 1-selective antibodies were generated against phosphor-serine conatiannig peptide taken form the region corresponding to aa    . Phospho-Slingshot1 isoform1 (PSSH1)-antibodies are affinity purified over immobilized antigen based chromatography, the pan and non-phospho antibodies were then removed and purified phsopho-specific antibodies were stabilized in antibody stabilization buffer. FabGennix Int. Inc. will also provide limited quantities of antigenic blocking phosphor-peptide for PSISHT-140AP antibody. Antibodies to PSSH1 (PSISHT-140AP) will label ~126kDa protein in Western blot positive control samples for PSSH1 and not in non-phospho protein PC-SISHTsamples.   FabGennix Inc. will also conjugate antibodies with secondary enzymes (alk-Pase or HRP) or fluorescent probes upon request at nominal cost.

Immunogen:            Synthetic peptides corresponding to unique epitope on SISHT.  The peptide sequence (LKR SHS(p) LAK LGS L) was unique to SISHT.  The PSISHT peptide was covalently modified post-synthetically covalently modified to achieve desired antigenicity. 

Concentration:         PSISHT -140AP:  IgG concentration 0.64-0.72 mg/ml in antibody stabilization buffer.

Applications:           Antibody PSISHT -140AP is ideal for WB and ELISA applications, other applications have not been tested.  These antibodies do not cross react to human lactoferrin, bovine lactoferrin or bovine serum albumin.  The species cross reactivity for these antibodies have not been examined fully.  The dilutions for this antibody is for reference only, investigators are expected to determine the optimal conditions for specific assay.  WB: > 1:500; IMM & i.p pull-down assays: n.d; IHC n.d.  ELISA <1:10,000.  Application of this antibody in protocols not listed here does not necessarily exclude its use in such procedures.

Reactivity:                This antibody detects a single band of approximately 126kDa in PC- PSISHT samples.  The antibody does not cross react to non-phospho-SISHT protein in PC-SISHT samples. 

Protocols:                Standard protocol for various applications (WB; IMM and IHC) of this antibody is provided with the product specification sheet, however, FabGennix Inc., strongly recommends investigators to optimize conditions for use of this antibody in their laboratories.  

Form/Storage:          The antiserum is supplied in antibody stabilization buffer.  The affinity-purified antibodies are isolated on immobilized antigen-affinity column and supplied as stabilized product.  Store at -20^oC for long-term storage.  FabGennix Inc. does not recommend storage of very dilute antibody solutions unless they are prepared in specially formulated multi use antibody dilution buffer (Cat # DiluOBuffer).  Working solutions of antibodies in DiluOBuffer should be filtered through 0.45um filter after every use for long-term storage.

Notes:  Briefly centrifuge to collect liquid, heat or boil PC-PSISHT tube for 1-2 minutes to dissolve any precipitate before use.  This product is “ready-to-use” for electrophoresis. After thawing store at room temperature, Repeated freezing and thawing may result in appearance of higher MW immunoreactive bands.   

New Reagents:  Now you can recycle your western blots (nitrocellulose, supported membranes and PVFD membranes) by using our StripObuffer (Cat FGI-1989).  Each stripping is guaranteed to give better signal (up to 8 stripping).  No strong pungent smell of reducing agents or heating required.  Block in 5X diluObuffer and you are ready for blotting with a new antibody

 

 

For research use only, not for diagnostic or therapeutic use.