Screening and identification of proteins interacting with nucleostemin

Figure 1 Identification of the recombinant clone of NS and its expression in yeast cell. A: Analysis of pGBKT7-NS with restriction enzyme digestion. M: DNA markers; lane 1: undigested pGBKT7-NS; lane 2: digestion of pGBKT7-NS with BamHI/XhoI and 1 680-bp fragment was released; B: Western blotting analysis of NS expression by pGBKT7-NS in yeast. Total protein from yeast AH109 transferred with pGBKT7-NS was subjected to Western blot and the NS mAb was used to detect the NS protein. Lane 1: total proteins from pGBKT7-NS transferred AH109; lane 2: total proteins from pGBKT7 transferred AH109.

Figure 2 Restriction enzyme analysis of recombinant pcDNA3-NS and pcDNA3-myc-PPP2R5A. M: DNA markers; lane 1: undigested pCDNA3-NS; lane 2: digestion of pCDNA3-NS by BamHI/XhoI and 1 680-bp fragment was released; lane 3: undigested pcDNA3-myc-PPP2R5A; lane 4: digestion of pcDNA3-myc-PPP2R5A with BamHI/XhoI and 2 300 bp fragment was released.

Figure 3 Co-immunoprecipitation of protein NS and PPP2R5A. Total proteins from cells co-transferred with plasmids pCDNA3-NS and pcDNA3-myc-PPP2R5A were used for immunoprecipitation with antibody against NS or c-myc and antibody against c-myc or NS was used in Western blot. Mouse IgG was used in immunoprecipitation as negative control.