pretransformed mouse adult testis matchmaker cdna library  (TaKaRa)

Journal: Journal of Biological Chemistry

Article Title: Mouse GGN1 and GGN3, Two Germ Cell-specific Proteins from the Single Gene Ggn, Interact with Mouse POG and Play a Role in Spermatogenesis

doi: 10.1074/jbc.m211023200

Figure Lengend Snippet: FIG. 3. Interaction between POG and GGN1/GGN3. A, interac- tion between POG and GGN3 in yeast. Amino acids 182–274 of POG were enough to mediate the interaction with GGN3. The interaction still existed after switching the Pog and Ggn3 cDNA between the two vectors. B and C, co-immunoprecipitation experiments to confirm the interaction between POG and GGN1 in mammalian cells. B, GAL4- tagged POG and MYC-tagged GGN1 expressing plasmids were trans- fected into COS-1 cells. A specific band of 40 kDa (possibly the N terminus-degraded protein generated in the process of immunoprecipi- tation; since the Myc tag was in the C terminus of the protein, the degradation of the N terminus did not interfere with the co-immuno- precipitation) could be seen in co-transfection immunoprecipitation but not in single transfection immunoprecipitation. C, Myc-tagged GGN1 and Xpress-tagged POG-expressing plasmids were transfected into COS-1 cells. Xpress-tagged POG co-immunoprecipitated with Myc- tagged GGN1. D, mapping the region critical for GGN3 homodimeriza- tion. Amino acids 66–132 of GGN3 were able to mediate the homodimerization.

Article Snippet: Since the presence of the POG C-terminal PHD domain in the bait caused autoactivation of the reporter gene upon transformation into AH109, the PHD domain-coding region was deleted from the bait construct and used to screen a pretransformed mouse adult testis cDNA library (Clontech).

Techniques: Immunoprecipitation, Expressing, Generated, Cotransfection, Transfection