به منظور بررسي جايگاه درون سلولي آنزيم COS_6 به لاين سلولي HSKI-1 انتقال ژن

Transfer of hSKI-1V-5 cDNA in COS-7 Cells to Determine Subcellular Localization

In contrast to mammalian Precursor Convertases (PCs), Site-1- Protease /Subtilisi n-Kexin-Isozyme-1 (S1P/SKI-1) cleaves its substrates at non-basic residues in the proposed sequence of: (Arg/Lys)-X-(hydrophobic)-(L,T) -X. SIP/SKI-1 seems to be present in the endoplasmic reticulum (ER), Golgi apparatus and secretory vesicles and probably primarily active in the Golgi apparatus. In the present study, we have determined the intracellular localization of the enzyme by immuno-cytochemistry. Briefly, COS-7 cells were transfected by a vector containing Si.P/SKI-1-V5 cDNA. Since there is an EGFP cDNA (as a reporter) in the vector, the level of transfection and the percentage of the transfected cells were determined using fluorescence microscopy. for evaluating the expression of exogenous S1P/SKI-1-V5, total RNA from transfected cells was extracted using RNX (Plus) solution. After spectrophotometry and agarose gel electrophoresis of extracted RNA, reverse transcription was performed by oligo dT primer and M-MLV enzyme. cDNA was amplified in PCR reaction for SIP/SKI-1-V5 and the product of PCR was PAGE electrophoresed. Finally, immunostaining on monolayer of transfected cells was performed using anti-V5 antibody. The staining pattern, in immuno-cytochemistry of transfected cells, revealed that S1P/SKI-1-V5 exists in compartments with characteristics of ER and Golgi. However, the highest level of signal was observed around the nucleus. The staining pattern confirms the previous report and suggests that S1P/SKI-1 resides primarily in ER and Golgi compartments.