ovary cdna library  (TaKaRa)

Journal: Nuclear Receptor

Article Title: PLZF is a negative regulator of retinoic acid receptor transcriptional activity

doi: 10.1186/1478-1336-1-6

Figure Lengend Snippet: Structure and properties of the bait RAR mutant and of one of the identified preys, PLZF. A) Schematic representation of the nuclear receptor RARα and structural localization of the two mutations K262A and K244A. These mutations weaken the interaction with the corepressor SMRT and abolish the interaction with the coactivator SRC-1, as visualized by GST pull-down assays (insert). B) Structure of the transcription factor PLZF identified by the two-hybrid screening of an ovary cDNA library with pLex12-RAR K244A-K262A used as a bait.

Article Snippet: An ovary cDNA library (in pACT2 vector, Clontech) was screened using the L40a yeast strain transformed with the pLex10-RARK244A-K262A vector, essentially as described in [ ].

Techniques: Mutagenesis, Two Hybrid Screening, cDNA Library Assay

Journal: Cells

Article Title: Homeoprotein Msx1-PIASy Interaction Inhibits Angiogenesis

doi: 10.3390/cells9081854

Figure Lengend Snippet: Homeoprotein Msx1 interacts with PIASy. ( a ) Identification of PIASy as Msx1-interacting protein by yeast 2-hybrid (Y2H) screening. The amino acid sequence of PIASy is presented using single letter abbreviations. The underlined amino acid sequence is the translated PIASy protein isolated from the yeast two-hybrid (Y2H) assay. Msx1 and PIASy cDNA constructs were introduced into EGY48 yeast host strains to test for protein-protein interactions within the Y2H analysis system. Transformants were examined for their ability to grow on a medium lacking leucine at 30 °C ( b , upper panel) and for β-galactosidase expression (b, bottom panel). ( c ) Biological interaction between cDNA constructs for three truncated Msx1 (Met 1 -Leu 160 , Arg 161 -Gln 225 , and Glu 226 -Thr 297 ) and three deleted PIASy (Met 1 -Arg 180 , Glu 181 -Ser 414 , and Cys 415 -Cys 510 ) fusion proteins in the Y2H analysis system. ( d ) Interaction of Msx1 and PIASy in mammalian cells. HEK 293 cells were infected with Ad-mock or Ad-Msx1 and then transiently transfected with Flag-PIASy harboring a full-length PIASy. The whole cell lysates were used for immunoprecipitation with anti-IgG or anti-Msx1 antibody, followed by immunoblotting with anti-Flag antibody. Expression of Msx1 was confirmed by immunoblotting with anti-Msx1 antibody. ( e ) Nuclear co-localization of Msx1 and PIASy in human ovarian cancer cells. Human ovarian cancer 2774 cells were co-transfected with pEGFP-Msx1 expressing a full-length Msx1 and Flag-PIASy expressing a full-length PIASy and then subjected to indirect immunofluorescence staining with Alexa Fluor 568-conjugated secondary antibody. The subcellular co-localization of Msx1-PIASy was examined using confocal microscopy. The co-localization of EGFP-Msx1 and Flag-PIASy is shown in the merged images. Scale bar, 10 μm.

Article Snippet: The pGilda/LexA-human Msx1 fusion protein was constructed and used to screen binding proteins from a human ovary cDNA library (Clontech).

Techniques: Sequencing, Isolation, Y2H Assay, Construct, Protein-Protein interactions, Expressing, Infection, Transfection, Immunoprecipitation, Western Blot, Immunofluorescence, Staining, Confocal Microscopy