Journal: PLoS ONE

Article Title: FE65 Binds Teashirt, Inhibiting Expression of the Primate-Specific Caspase-4

doi: 10.1371/journal.pone.0005071

Figure Lengend Snippet: Clones identified in yeast two hybrid screens

Article Snippet: After validating that the PTB1 domain did not, by itself, activate reporter gene expression, adult mouse and fetal human brain cDNA libraries were screened (Clontech, Mountain View, CA) resulting in 41 and 38 positive clones respectively.

Techniques: Clone Assay, cDNA Library Assay

Journal: BMC Cancer

Article Title: The bone morphogenetic protein antagonist gremlin 1 is overexpressed in human cancers and interacts with YWHAH protein

doi: 10.1186/1471-2407-6-74

Figure Lengend Snippet: Identification and expression level of PIG-2 . A ) Comparison gene expression profiles by DDRT-PCR from total RNA isolated from normal cervical tissue, primary cervical cancer, cervical cancer tissue metastatic to lymph node and from CasKi and CUMC-6 cervical cancer cell lines. Differential display was carried out 5' arbitrary primer H-AP28 (5' -AAGCTTACGATGC-3') and 3' H-T 11 C (5' -AAGCTTTTTTTTTTTC-3'). The PCR products were resolved by electrophoresis. CC282 is the name of the partial PIG-2 gene product. The arrow identifies the location relative to other PCR products. ( B ) Total RNAs were isolated from normal cervical tissue, primary cervical cancer, cervical cancer tissue metastatic to lymph node and from CasKi andCUMC-6 cervical cancer cell lines. Blot was hybridized with the randomly primed [ 32 P]-labeled PIG-2 partial cDNA probe (the CC282 fragment). Human β-actin cDNA was used as a control probe (lower panel).

Article Snippet: Yeast cells expressing the LexA-PIG-2 were transformed with a human fetal brain cDNA library (Invitrogen) that expresses B42AD fusion proteins.

Techniques: Expressing, Isolation, Electrophoresis, Labeling

Journal: BMC Cancer

Article Title: The bone morphogenetic protein antagonist gremlin 1 is overexpressed in human cancers and interacts with YWHAH protein

doi: 10.1186/1471-2407-6-74

Figure Lengend Snippet: Expression of PIG-2 in human tissues by Northern analyses and immunohistochemical studies. Northern blotting analyses were performed to determine the expression of PIG-2 in different human tissues. Normal 12 lane multiple tissue northern blot ( A ) or human cancer cell line multiple northern-blot purchased from Clontech ( B ) was probed with a radioactively labeled CC282 partial cDNA (upper panel) or human β-actin cDNA control probe provided by Clontech (lower panel). ( C ) Total RNAs were isolated from normal lung tissue, primary lung cancer and from NCI-H441, NCI-H157 and NCI-H2009 lung cancer cell lines. Blot was hybridized with the randomly primed [ 32 P]-labeled PIG-2 partial cDNA probe (the CC282 fragment). Human β-actin cDNA was used as a control probe (lower panel). ( D ) Comparison of PIG-2 mRNA expression in human tumor tissues and their corresponding normal counterparts (upper panel). Total RNAs were extracted from fresh human normal and cancer tissues. The same blot was probed with β-actin as a loading control (lower panel). ( E-I ) Immunohistochemical staining for PIG-2 expression of human normal muscle ( E ), leiomyosarcoma ( F ), normal colon tissue ( G ), colon cancer ( H ), and pancrease cancer tissue ( I ), Original magnification, × 100.

Article Snippet: Yeast cells expressing the LexA-PIG-2 were transformed with a human fetal brain cDNA library (Invitrogen) that expresses B42AD fusion proteins.

Techniques: Expressing, Northern Blot, Immunohistochemical staining, Labeling, Isolation, Staining

Journal: BMC Cancer

Article Title: The bone morphogenetic protein antagonist gremlin 1 is overexpressed in human cancers and interacts with YWHAH protein

doi: 10.1186/1471-2407-6-74

Figure Lengend Snippet: Association of PIG-2 and YWHAH . ( A ) Mapping of PIG-2 binding domain of YWHAH. Five GST fusion constructs, Full-YWHAHp1-247, YWHAHp1-100, YWHAHp1–80, YWHAHp1–60 and YWHAHp81–247 were prepared using YWHAH cDNA as a template DNA. The GST pull-down assay showed that Three GST-YWHAH constructs, Full-YWHAHp1–247, YWHAHp1–100 and YWHAHp1–80 bind YWHAH, but not YWHAHp1–60 and YWHAHp60–247. The YWHAH binding site for PIG-2 was delineated to be residues 61–80 (black box). ( B ) Mapping of YWHAH binding domain of PIG-2. Four GST fusion constructs, Full-PIG-2p1-184, PIG-2p1-144, PIG-2p1-100 and PIG-2p1-67 were prepared using PIG-2 cDNA as a template DNA. The GST pull-down assay showed all constructs, The PIG-2 binding site for YWHAH was delineated to be residues 1–67. Gray box is DAN domain. ( C ) Schematic diagram shows X-ray crystallography of YWHAH protein. PIG-2 protein binding site was indicated by arrow.

Article Snippet: Yeast cells expressing the LexA-PIG-2 were transformed with a human fetal brain cDNA library (Invitrogen) that expresses B42AD fusion proteins.

Techniques: Binding Assay, Construct, Pull Down Assay, Protein Binding

Journal: PLoS ONE

Article Title: Direct Interaction of Selenoprotein R with Clusterin and Its Possible Role in Alzheimer’s Disease

doi: 10.1371/journal.pone.0066384

Figure Lengend Snippet: Plasmids carrying on the fetal brain cDNA library were co-transformed into the NpGBKT7- SelR′ -containing yeast and screened by the selection plate for the blue colonies (A). The interaction between SelR′ and Clu was verified by re-transformation of the plasmids NpGBKT7- SelR′ and pACT2- Clu into either AH109 (B) or Y2HGold (C) yeast cells. Yeast cells in B (1–3) were transformed with single NpGBKT7- SelR′ , NpGBKT7- SelR′ plus pACT2, and NpGBKT7- SelR′ plus pACT2- Clu plasmids, respectively. Yeast cells in C, D, E were transformed with NpGBKT7- SelR′ plus pACT2- Clu , pGBKT7- p53 plus pADT7- T (positive control), and pGBKT7- Lam plus pADT7- T (negative control), respectively, followed by the selection on SD/−Leu/−Trp/X-α-Gal/Aba plates.

Article Snippet: Matchmaker™ Gold yeast two-hybrid system, yeast strains Y2HGold and AH109, plasmids pACT2 and NpGBKT7, and human fetal brain cDNA library (using pACT2 as the vector) were purchased from Clontech Laboratories (Mountainview, CA, USA).

Techniques: cDNA Library Assay, Transformation Assay, Selection, Positive Control, Negative Control

Journal: PLoS ONE

Article Title: GRG5/AES Interacts with T-Cell Factor 4 (TCF4) and Downregulates Wnt Signaling in Human Cells and Zebrafish Embryos

doi: 10.1371/journal.pone.0067694

Figure Lengend Snippet: The AH109 yeast strain was co-transformed with the indicated constructs (central panel). Transformants were selected in medium lacking tryptophan and leucine (left panel) and activation of a Gal4-dependent ADE2 gene was examined by growth on selective plates additionally lacking adenine (right panel). Yeast cells containing both TCF4 and GRG5/AES grew in the selective media. pLAM and pACT2 plasmids were used as bait and prey negative controls respectively, while the β-catenin prey plasmid constitutes a positive control for the interaction assays.

Article Snippet: A commercial human fetal brain cDNA library in pACT2 (Clontech), was screened by PEG/LiAcetate co-transformation [ ] with the bait plasmid pAS2-TCF4 into the AH109 yeast reporter strain (which contains integrated copies of ADE2 and HIS3 reporter genes under control of Gal4-dependent promoters).

Techniques: Transformation Assay, Construct, Activation Assay, Plasmid Preparation, Positive Control