This Mouse Reticulocalbin 3/RCN3 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of Reticulocalbin 3/RCN3 protein (Cat: 51928-M08H) from the overexpression lysate was verified.
A DNA sequence encoding the mouse Rcn3 (NP_080831.2) (Met1-Leu328) was expressed with a polyhistidine tag at the C-terminus.
The recombinant mouse Rcn3 consists of 319 amino acids and predicts a molecular mass of 36.9 kDa.
Cell lysate was prepared by homogenization of the over-expressed cells in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors (Sigma). Cell debris was removed by centrifugation. Protein concentration was determined by Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The cell lysate was boiled for 5 min in 1 x SDS loading buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol, and lyophilized.
RCN3 belongs to the CREC family which contains multiple EF-hand Ca2+-binding proteins localized to the secretory pathway. RCN3 sequence is characterized by the presence of five Arg-Xaa-Xaa-Arg motifs, which represents the target sequence of subtilisin-like proprotein convertases(SPCs). SPCs are a family of seven structurally related serine endoproteases that are involved in the proteolytic activation of proproteins.RCN3 is transiently associated with proPACE4, but not with mature PACE4. Inhibition of PACE4 maturation by a Ca2+ ionophore resulted in accumulation of the proPACE4-RCN-3 complex in cells. It has been proposed that elective and transient association of RCN3 with the precursor of PACE4 plays an important role in the biosynthesis of PACE4.
reticulocalbin 3, EF-hand calcium binding domain
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