• Soo Jin Park, Byung Ha Lee, and Dae Joong Kim.
• Clinical Research Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Gangnam-Gu, Seoul, Korea.
• Yonsei Med. J. 2009 Apr 30; 50 (2): 273279273-9.

PurposeAs a membrane protein at the insertion site of the slit diaphragm (SD) complex in podocyte foot processes, podocin has been reported to act as a scaffolding protein required to maintain or regulate the structural integrity of the SD. In order to identify proteins that associate or interact with podocin, we screened a mouse kidney complementary DNA (cDNA) library using a yeast 2-hybrid system.Materials And Methods1) The full-length cDNA of podocin from the mouse kidney was amplified by Polymerase Chain Reaction (PCR), 2) The PCR product was cloned into a pGBKT7 vector, pGBKT7-podocin, 3) After the pGBKT7-podocin was transformed into AH109, the AH109/pGBKT7-podocin product was obtained, 4) The mouse kidney cDNA library was transformed into the AH109/pGBKT7-podocin and screened by selection steps, 5) Next, twelve clones were cultured and isolated, 6) The yeast-purified plasmids were transformed into Escherichia coli (E. coli) by heat shock, and 7) To identify the activation domain (AD)/library inserts, we digested them with Him III, and the fragments were then sequenced.Results12 positive clones that interacted with podocin were obtained by screening a mouse kidney cDNA library using pGBKT7-podocin. Among them, only 4 clones were found to function at the podocyte where podocin is present.ConclusionAdditional studies are needed to clarify the role and interaction with podocin and candidates.

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