tet off hela s3 cells  (TaKaRa)

Journal:

Article Title: U2AF modulates poly(A) length control by the poly(A)-limiting element

doi: 10.1093/nar/gkg823

Figure Lengend Snippet: Impact of U2AF65 and PAP-interacting domain deletions on PLE regulation of poly(A) tail length. (A) Plasmids expressing wild-type U2AF65 or U2AF65 deleted for amino acids 17–27 (Δ17–27) or 17–47 (Δ17–47) were transfected into CHO cells and expression was analyzed by western blot using a polyclonal antibody to human U2AF65. In lane 1 cells were transfected with pcDNA3 alone. (B) HeLa S3 (Tet-Off) cells were transfected in tetracycline-containing medium with the indicated U2AF65 constructs plus tetracycline-regulated plasmids bearing human β-globin reporter genes that lack (SPA) or contain a PLE (PLE). Poly(A) tail length was analyzed by RT–PCR on RNA isolated 30 h after transfection and 6 h after removing tetracycline from the medium. The center lane (lane 5) contains a marker of Hinf φX174 DNA fragments. (C) The graphing function of the ImageQuant™ program was used to determine the distribution of radioactivity in each of the lanes in (B). Note that the scales for control and PLE-containing mRNAs are different.

Article Snippet: Tet-Off HeLa S3 cells and CHO cells were purchased from Clontech and maintained in DMEM with 10% FBS, 4 mM glutamine and 100 µg/ml G418 (Invitrogen).

Techniques: Expressing, Transfection, Western Blot, Construct, Reverse Transcription Polymerase Chain Reaction, Isolation, Marker, Radioactivity

Journal:

Article Title: U2AF modulates poly(A) length control by the poly(A)-limiting element

doi: 10.1093/nar/gkg823

Figure Lengend Snippet: Impact of overexpressing U2AF65 on polyadenylation of mRNA with the C14G mutation. HeLa S3 (Tet-Off™) cells were transfected as in Figure ​Figure66 with tetracycline-regulated plasmids bearing the PLE or C14G element in the last exon of the β-globin reporter gene plus empty vector (pcDNA) and CMV-driven plasmids expressing full-length U2AF65 or the Δ17–47 mutant form of U2AF lacking the PAP-interacting domain. (A) Poly(A) tail length was determined by RT–PCR as in Figure ​Figure66 and equal amounts of radiolabeled products were applied to the gel. Lane 1 (M) contains a marker of Hinf φX174 DNA fragments. (B) The graphing function of the ImageQuant™ program was used to determine the distribution of radioactivity in each of the lanes for PLE-containing mRNA. The dashed line corresponds to cells transfected with the Δ17–47 form of U2AF65.

Article Snippet: Tet-Off HeLa S3 cells and CHO cells were purchased from Clontech and maintained in DMEM with 10% FBS, 4 mM glutamine and 100 µg/ml G418 (Invitrogen).

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction, Marker, Radioactivity

Journal:

Article Title: Structural and Functional Conservation of the NuA4 Histone Acetyltransferase Complex from Yeast to Humans

doi: 10.1128/MCB.24.5.1884-1896.2004

Figure Lengend Snippet: SANT domain-containing protein DMAP1 is a stable subunit of the human NuA4 complex. (A) Amino acid sequence alignment between yeast NuA4 subunit Eaf2, human DMAP1, and a Drosophila homologous protein (dEaf2, accession number AAF57436). The conserved SANT domain region is indicated. (B) DMAP1 associates with the same set of proteins as Tip60. Nuclear extracts from FLAG-Tip60-TAP, FLAG-DMAP1-TAP, or mock-transduced HeLa S3 cells were partially purified over IgG-Sepharose beads, and the TEV eluate was analyzed by Western blotting as in Fig. ​Fig.1B.1B. (C) Triple affinity purification of DMAP1 identifies distinct non-HAT complex(es). The DMAP1-containing complexes were purified as in Fig. ​Fig.22 and analyzed on a gel by quantitative Sypro Ruby staining. Proteins identified by Western analysis or tandem mass spectrometry are labeled on the right. New proteins not identified in the Tip60 purification are in italics. The positions of MRG15 and Tip60 based on Western analysis of the previous fraction are given in parentheses. The asterisk indicates a nonspecific band. A significant amount of GAS41 protein was also detected but was run out of the gel presented here (see Western signal in panel B). The numbers of specific peptide sequences obtained by tandem mass spectrometry analysis performed as described for Fig. ​Fig.22 are as follows: 6 for TRRAP, 8 for p400/hDomino, 7 for SRCAP (2,971 aa), 4 for Brd8, 13 for DMAP1, 4 for YL-1 (364 aa), 10 for RUVBL1, 14 for RUVBL2, 5 for BAF53a, and 5 for actin.

Article Snippet: MCF7 and HeLa S3 tet-off cell lines were purchased from Clontech and cultured in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum.

Techniques: Sequencing, Purification, Western Blot, Affinity Purification, Staining, Mass Spectrometry, Labeling