Journal: Journal of Biological Chemistry

Article Title: Interplay between HIV-1 Vpr and Sp1 Modulates p21WAF1 Gene Expression in Human Astrocytes

doi: 10.1074/jbc.m403792200

Figure Lengend Snippet: FIG. 2. Transcription activation of the p21 promoter by Vpr in the presence of Sp1. A, U-87 MG cells were transfected with 0.5 g of the reporter p21 luciferase plasmids alone or in combination with 2.5 g of either Vpr or Sp1 expression plasmids as described in Fig. 1. The data represent the mean value of at least three separate transfection experiments. B, SL2 cells were transfected with 0.5 g of the p21-luc promoter constructs of either pPac-Sp1 or CMV-Vpr expression plasmids. Empty pCEP-4 or pPac-0 vectors were added in all transfection mixtures to normalize the amounts of the DNA in each reaction. Cell extracts were prepared 48 h after transfection, and luciferase activity was determined. C, bandshift assay was performed by using 32P-labeled, 20-bp double-stranded synthetic DNA fragments containing the wild type GC-rich motif of the p21 promoter (89/70) or its mutant with no binding site to Sp1. Approximately 105 cpm labeled DNA probes were incubated with 10 g of nuclear extracts prepared from cells transfected with either Sp1, Vpr, or in combination, as indicated above each lane. Supershift experiments were performed with 10 g of nuclear proteins in the presence of antibodies directed against Sp1 as indicated. C1 demonstrates complexes formed upon addition of nuclear extracts. NS (nonspecific) points to the position of a nonspecific complex, which was detected in some but not all reactions. C0 points at the complex supershifted by Sp1 antibodies. p indicates free probe.

Article Snippet: For supershift assays, antibodies directed against Sp1 or p53 (Santa Cruz Biotechnology) were mixed with nuclear proteins for 1 h at 4 °C prior to addition of the probe.

Techniques: Activation Assay, Transfection, Luciferase, Expressing, Construct, Activity Assay, Labeling, Mutagenesis, Binding Assay, Incubation

Journal: OncoTargets and Therapy

Article Title:

1,25-Dihydroxyvitamin D₃ affects gastric cancer progression by repressing BMP3 promoter methylation

doi: 10.2147/ott.s195642

Figure Lengend Snippet: Figure 4 1,25(OH)2 D3 inhibits BMP3 promoter methylation required with VDR influencing. Notes: (A) BMP3 expression in gastric noncancer and cancer cells lines was detected by Western blot. (B) BMP3 expression of gastric cancer cell line SGC-7901 was detected by Western blot after treating with gradient concentration of 5-AZ (0, 0.1, 0.2, 0.3, 0.4, 0.5 mM). (C) BMP3 expression of three gastric cancer cell lines (BGC- 823, SGC-7901, and MKN-28) and normal line GES-1 was detected by Western blot with or without 1,25(OH)2 D3 (0.1 μM) treatment. Data are represented as mean±SD (*P0.05 and **P0.01). (D) Gel EMSA was performed using the nuclear extract from SGC-7901 cells. A labeled wild-type probe containing the VDR consensus binding sequence (5′-GGGTGGCTAAGGGCA-3′) of the BMP3 promoter was used to test VDR binding. The supershift band and bindshift band are indicated by arrows. Abbreviations: 1,25(OH)2 D3, 1,25-dihydroxyvitamin D3; VDR, vitamin D receptor; 5-AZ, 5-aza-2′ deoxycytidine; EMSA, electrophoretic mobility shift assay.

Article Snippet: For supershift experiments, VDR (SC-13133; Santa Cruz Biotechnology) his tag and anti-his tag (LT0424; LifeTein, South Plainfield, NJ, USA) were added to the reaction solution 30 minutes prior to the addition of the probes.

Techniques: Methylation, Expressing, Western Blot, Concentration Assay, Labeling, Binding Assay, Sequencing, Electrophoretic Mobility Shift Assay

Journal: Oncogene

Article Title: Polyomavirus large T antigen-dependent DNA amplification.

doi: 10.1038/sj.onc.1200904

Figure Lengend Snippet: Figure 1 (a) Induction of wild type or mutated PyLT in stably transfected REF52 cells by addition of 1 mmol/l of dexametha- sone. Extracts were prepared from logarithmically growing cells, proteins (20 ug total protein per lane) were separated in 7.5% polyacrylamide gels and PyLT was stained with anti PyLT antibody. (b) GST pull down experiment showing that GST-pRB and GST-p107 bind wild type but not mutated PyLT. The experiment was carried out as described previously (Karlseder et al., 1996) using 150 mg of total protein of extracts from wild type or mutated PyLT-producing cells

Article Snippet: E2F complexes containing p107 or p130 were supershifted by incubating the reaction with the p107 speci®c monoclonal antibody SD15 (Dyson et al., 1992) or with the p130 speci®c antibody C-20 (Santa Cruz).

Techniques: Stable Transfection, Transfection, Staining

Journal: Oncogene

Article Title: Polyomavirus large T antigen-dependent DNA amplification.

doi: 10.1038/sj.onc.1200904

Figure Lengend Snippet: Figure 6 (a) PyLT synthesized in logarithmically growing cells does not dissociate complexes between E2F and pocket protein. Mobility shift experiments were carried out as described in Materials and methods using the E2F motif of the murine TK promoter as labeled oligonucleotide. Proteins in extracts of growing cells were used. REF52 cells did not produce a viral protein and represent the control, COP8 cells are polyomavirus transformed murine C127 cells which produce large amounts of all three tumor antigens. SVMK cells are SV40 transformed mouse kidney cells, producing SV40 large and small T antigen. SD15 is an anti-p107 antibody giving rise to a supershift of p107-containing complexes. (b) LT dissociates complexes present in quiescent cells. These complexes contain the pocket protein p130 as they are supershifted by antibody speci®c for this protein (C-20) while no supershift is obtained with anti-p107 antibody

Article Snippet: E2F complexes containing p107 or p130 were supershifted by incubating the reaction with the p107 speci®c monoclonal antibody SD15 (Dyson et al., 1992) or with the p130 speci®c antibody C-20 (Santa Cruz).

Techniques: Synthesized, Mobility Shift, Labeling, Control, Transformation Assay