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Mouse KIRREL3 / NEPH2 Human Cells Transfected Lysate (positive control) (denatured)

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KIRREL3/NEPH2Transfected / Overexpression Cell Lysate Product Information
Product Description:Human Cells transfected lysate in which Mouse KIRREL3 has been over-expressed. The whole cell lysate is provided in 1X Sample Buffer (1X modified RIPA buffer+1X SDS sample buffer).
Preparation Method:Cell lysate was prepared by homogenization in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors (Sigma). Cell debris was removed by centrifugation. Protein concentration was determined with Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The cell lysate was boiled for 5 minutes in 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol, and lyophilized.
Lysis Buffer:Modified RIPA Lysis Buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, 1% Sodium deoxycholate, 1mM PMSF
Quality Control Testing:12.5% SDS-PAGE Stained with Coomassie Blue
Stability:Samples are stable for up to twelve months from date of receipt at -80℃
Recommend Usage:1. Centrifuge the tube for a few seconds and ensure the pellet at the bottom of the tube. 2. Re-dissolve the pellet using 200μL pure water and boiled for 2-5 min. 3. Store it at -80℃. Recommend to aliquot the cell lysate into smaller quantities for optimal storage. Avoid repeated freeze-thaw cycles. Notes:The lysate is ready to load on SDS-PAGE for Western blot application. If dissociating conditions are required, add reducing agent prior to heating.
Storage Buffer:In modified RIPA Lysis Buffer
Storage Instruction:Store at -80℃. Aliquot to avoid repeated freezing and thawing
Application notes:WB: Use at an assay dependent dilution.
Not yet tested in other applications.
Optimal dilutions/concentrations should be determined by the end user.

Kin of IRRE-like protein 3 (KIRREL3) also known as nin of irregular chiasm-like protein 3 or nephrin-like protein 2 (NEPH2) is a member of the nephrin-like protein family of transmembrane proteins, which includes NEPH1 (KIRREL) and NEPH3 (KIRREL2). KIRREL3/NEPH2 is expressed in fetalv and adult brain, and also in podocytes of kidney glomeruli. The cytoplasmic domains of KIRREL3/NEPH2 interact with the C-terminus of podocin, also expressed in the podocytes, cells involved in ensuring size- and charge-selective ultrafiltration. Mutations in KIRREL3/NEPH2 are associated with mental retardation autosomal dominant type 4. KIRREL3/NEPH2 expression is turned on in migrating nucleogenesis of the pontine nucleus (PN) neurons only after they enter the presumptive nuclear region. KIRREL3/NEPH2 knockdown disrupted the nuclear organization of PN presumably by changing the migratory behavior of PN neurons inside the nuclear region. Moreover, overexpression of the cytoplasmic region of KIRREL3, which can sequester intracellular signaling of endogenous KIRREL3, resulted in similar phenotypes. Overall, these results suggest KIRREL3 is involved in the nucleogenesis of the PN through the control of neuronal migration inside the nucleus.

  • Bhalla K, et al. (2008) Alterations in CDH15 and KIRREL3 in patients with mild to severe intellectual disability. Am J Hum Genet. 83(6): 703-13.
  • Gerke P, et al. (2005) NEPH2 is located at the glomerular slit diaphragm, interacts with nephrin and is cleaved from podocytes by metalloproteinases. J Am Soc Nephrol. 16(6): 1693-702.
  • Gerke P, et al. (2006) Neuronal expression and interaction with the synaptic protein CASK suggest a role for Neph1 and Neph2 in synaptogenesis. J Comp Neurol. 498(4): 466-75.
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