Journal: Biochimica et biophysica acta

Article Title: Inhibition of Fas expression by RNAi modulates 5-fluorouracil-induced apoptosis in HCT116 cells expressing wild-type p53.

doi: 10.1016/j.bbadis.2006.09.005

Figure Lengend Snippet: Fig. 3. 5-FU increases p53 and Fas expression in p53-proficient cells. HCT116 cells were exposed to 8 μM 5-FU or no addition (control) for 48 h. Total proteins were extracted for immunoblot analysis. Representative immunoblots of p53 and Fas expression and corresponding protein levels. Representative immuno- blots of Bcl-2 and Bax are also shown. The results are expressed as mean±SEM arbitrary units of at least five independent experiments. §p<0.05 from controls. β-actin was used as a loading control.

Article Snippet: After blocking with 5%milk solution, the blots were incubated overnight at 4 °C with primary mouse monoclonal antibodies reactive to p53, Bax, Bcl-2, Fas (Santa Cruz Biotechnology), cytochrome c (BD Biosciences PharMingen, San Diego, CA) and cytochrome c oxidase subunit II (Molecular Probes, Eugene, OR), or primary rabbit polyclonal antibodies for PARP and caspase-3 (Santa Cruz Biotechnology).

Techniques: Expressing, Control, Western Blot

Journal: Biochimica et biophysica acta

Article Title: Inhibition of Fas expression by RNAi modulates 5-fluorouracil-induced apoptosis in HCT116 cells expressing wild-type p53.

doi: 10.1016/j.bbadis.2006.09.005

Figure Lengend Snippet: Fig. 5. Fas silencing reduces 5-FU-induced caspase activation in HCT116 cells. Cells were transfected with Fas siRNA or mock transfected at 24 h after plating. Twenty-four hours after transfection, 8 μM 5-FU or no addition (control) were included for additional 24 h. Cytosolic proteins were extracted for caspase activity and processing assays. (A) Caspase-8 and -9 activities. (B) Caspase-3 activity (left) and representative immunoblot of active caspase-3 (right). (C) Representative immunoblot of total p53 expression. The results are expressed as mean±SEM arbitrary units of at least three independent experiments. *p<0.001 and §p<0.05 from controls. β-actin was used to control for lane loading.

Article Snippet: After blocking with 5%milk solution, the blots were incubated overnight at 4 °C with primary mouse monoclonal antibodies reactive to p53, Bax, Bcl-2, Fas (Santa Cruz Biotechnology), cytochrome c (BD Biosciences PharMingen, San Diego, CA) and cytochrome c oxidase subunit II (Molecular Probes, Eugene, OR), or primary rabbit polyclonal antibodies for PARP and caspase-3 (Santa Cruz Biotechnology).

Techniques: Activation Assay, Transfection, Control, Activity Assay, Western Blot, Expressing

Journal: Biochimica et biophysica acta

Article Title: Inhibition of Fas expression by RNAi modulates 5-fluorouracil-induced apoptosis in HCT116 cells expressing wild-type p53.

doi: 10.1016/j.bbadis.2006.09.005

Figure Lengend Snippet: Fig. 6. 5-FU induces cytochrome c release and translocation of p53 and Bax to the mitochondria in HCT116 cells. Cells were exposed to 8 μM 5-FU or no addition (control) for 24 h. Mitochondrial proteins were extracted for immunoblot analysis. Representative immunoblots of p53, Bax and cytochrome c (Cyt c) and corresponding protein levels. The results are expressed as mean± SEM arbitrary units of at least three independent experiments. *p<0.001 and §p<0.05 from controls.

Article Snippet: After blocking with 5%milk solution, the blots were incubated overnight at 4 °C with primary mouse monoclonal antibodies reactive to p53, Bax, Bcl-2, Fas (Santa Cruz Biotechnology), cytochrome c (BD Biosciences PharMingen, San Diego, CA) and cytochrome c oxidase subunit II (Molecular Probes, Eugene, OR), or primary rabbit polyclonal antibodies for PARP and caspase-3 (Santa Cruz Biotechnology).

Techniques: Translocation Assay, Control, Western Blot

Journal: Oncogene

Article Title: Elevated urokinase-type plasminogen activator receptor expression in a colon cancer cell line is due to a constitutively activated extracellular signal-regulated kinase-1-dependent signaling cascade.

doi: 10.1038/sj.onc.1201098

Figure Lengend Snippet: Figure 1 Extracellular signal-regulated kinase 1 (ERK1) is constitutively activated in RKO cells. (a) Equal amounts of protein extract were subjected to SDS ± PAGE and the resolved proteins transferred to nitrocellulose. The ®lter was probed with an antibody, which crossreacts with human ERK1 and ERK2, and the bands visualized by ECL. Molecular weight markers are indicated to the left. (b) and (c) Equal amounts of extracted protein in the cell supernates were reacted with an anti-ERK1/ERK2 antibody and protein-A Sepharose, and the precipitate electrophoresed in a SDS ± PAGE gel containing myelin basic protein (MBP). The gel was washed with 2-propanol and treated sequentially with guanidine HCl and a buer containing Tween 40/2- mercaptoethanol. Phosphorylation of MBP was carried out by incubating the gel with [g32P]ATP. After incubation, the gel was washed and autoradiographed for 2 (b) or 16 h (c). (d) Cells (50 000) were plated on day 0 and proliferation rate assessed at the indicated times with MTT. The experiments were repeated twice or more

Article Snippet: The ®lter was blocked with 3% BSA and incubated with polyclonal antibodies reactive with human ERK1 and ERK2 (Santa Cruz Biotechnology #93) or with MEK1 and MEK2 (Transduction Laboratories #M17030).

Techniques: SDS Page, Molecular Weight, Phospho-proteomics, Incubation

Journal: Oncogene

Article Title: Elevated urokinase-type plasminogen activator receptor expression in a colon cancer cell line is due to a constitutively activated extracellular signal-regulated kinase-1-dependent signaling cascade.

doi: 10.1038/sj.onc.1201098

Figure Lengend Snippet: Figure 2 Repression of u-PAR promoter activity by the expression of an ERK-inactivating phosphatase or a dominant negative ERK1 expression vector. (a) and (b) RKO cells were co-transfected with a luciferase expression construct, a CAT reporter driven by the u-PAR promoter (u-PAR CAT), or a thymidine kinase minimal promoter (pBLCAT) ¯anked by 3 AP-1 tandem repeats (36AP-1 pBLCAT), with the indicated amounts of an expression vector (pSG5) encoding an active (CL100a pSG5) or inactive (CL100i pSG5) CL100 phosphatase. (c) RKO cells wre co-transfected as described for (a) with a u-PAR promoter-driven CAT reporter and an expression vector encoding a kinase-de®cient ERK1 (mt ERK1 pCEP4), a kinase-de®cient ERK2 (mt ERK2 pCEP4) or an amount of the empty expression vector (pCEP4) equimolar with 10 mg of ERK1 mt pCEP4. As controls, cells were transfected with a CAT reporter lacking a promoter (pSV0 CAT) or driven by the Rous sarcoma virus long terminal repeat (RSV CAT) or SV40 promoters (pSV2 CAT). [14C]-chloramphenicol conversions were determined with a 603 Betascope. The data are typical of at least duplicate experiments

Article Snippet: The ®lter was blocked with 3% BSA and incubated with polyclonal antibodies reactive with human ERK1 and ERK2 (Santa Cruz Biotechnology #93) or with MEK1 and MEK2 (Transduction Laboratories #M17030).

Techniques: Activity Assay, Expressing, Dominant Negative Mutation, Plasmid Preparation, Transfection, Luciferase, Construct, Virus

Journal: Oncogene

Article Title: Elevated urokinase-type plasminogen activator receptor expression in a colon cancer cell line is due to a constitutively activated extracellular signal-regulated kinase-1-dependent signaling cascade.

doi: 10.1038/sj.onc.1201098

Figure Lengend Snippet: Figure 3 The MEK1 inhibitor PD 098059 reduces u-PAR display in RKO cells. (a) and (b) Cells, at 90% con¯uency, were lysed with Triton X-100. For (a) the cell lysates (equal protein) were loaded onto a DE52-cellulose column. Aliquots of the ¯ow through were incubated with a 72 kDa kinase-inactive ERK1-GST fusion protein bound to glutathione agarose beads and [g-32P]ATP. The reaction was terminated with Laemmli sample buer and the mixture electrophoresed by SDS ± PAGE. Control represents a parallel incubation of the DE52-column equilibrating buer in the absence of cell extract. For (b) aliquots of the ¯ow through were subjected to Western blotting using a polyclonal antibody which crossreacts with MEK1 and MEK2. (c) Cells extracts prepared as for (a), or authentic MEK1, were incubated with (+), or without (7), 2 mg of an anti-MEK1/MEK2 antibody (a-MEK Ab) and Protein A-agarose beads. The immunoprecipitated material was washed and incubated with 1 mg of the ERK1-GST fusion protein and [g-32P]ATP at 308C. Reaction products were then resolved by SDS ± PAGE. (d) RKO cells were incubated for 4 days without (Control) or with the indicated concentrations of PD 098059 or an amount of DMSO (CARRIER) corresponding to the highest level of solvent used (0.05%). The cells were then treated for 3 min with a glycine buer and the acidic pH neutralized with a HEPES-containing buer (pH 7.4). The cells wre incubated at 48C for 2 h with 2.0 nM radioactive single chain urokinase (rscuPA) with, or without, a 100-fold excess of unlabeled ligand. The cells were subsequently washed and lysed with 1.0% Triton X-100. Speci®c binding was calculated by subtracting the amount of radioactivity bound in the presence of excess unlabeled ligand from that achieved in the absence and correcting for any dierences in cell number. The data represent the average values of two separate experiments. (e) RKO cells were treated for 48 h with the indicated concentration of PD 098059 or amounts of DMSO (CARRIER) corresponding to 10 mM of PD 098059. After this time, the cells were harvested, lysed and equal amounts of protein assayed for ERK activity as described in the legend to Figure 1

Article Snippet: The ®lter was blocked with 3% BSA and incubated with polyclonal antibodies reactive with human ERK1 and ERK2 (Santa Cruz Biotechnology #93) or with MEK1 and MEK2 (Transduction Laboratories #M17030).

Techniques: Incubation, SDS Page, Control, Western Blot, Immunoprecipitation, Solvent, Binding Assay, Radioactivity, Concentration Assay, Activity Assay

Journal:

Article Title: Functional Interactions between C/EBP, Sp1, and COUP-TF Regulate Human Immunodeficiency Virus Type 1 Gene Transcription in Human Brain Cells

doi:

Figure Lengend Snippet: Western blot analysis of nuclear proteins from human brain cells. (A and B) Nuclear protein extracts were prepared from oligodendroglioma TC-620 (lanes T), astrocytoma U373-MG (lanes U), and microglial (lanes M) cells. Proteins (10 μg) were subjected to SDS-PAGE (15% gel), transferred to a nitrocellulose membrane, and probed with antibodies of the C/EBP family. The same gel was cut in two and probed with anti-C/EBPβ (reactive with C/EBPβ and NF-IL6; Santa Cruz Biotechnology) (A) and anti-C/EBPγ (B). The signal was visualized with the Amersham ECL system. (C) Microglial cells were stimulated with IL-6 and TNF-α over a 24-h period. Nuclear proteins were subjected to SDS-PAGE (10% gel), transferred to a nitrocellulose membrane, and probed as in panel A. Positions of prestained molecular weight markers (Bio-Rad) are indicated on the left.

Article Snippet: The same gel was cut in two and probed with anti-C/EBPβ (reactive with C/EBPβ and NF-IL6; Santa Cruz Biotechnology) (A) and anti-C/EBPγ (B).

Techniques: Western Blot, SDS Page, Molecular Weight

Journal:

Article Title: Functional Interactions between C/EBP, Sp1, and COUP-TF Regulate Human Immunodeficiency Virus Type 1 Gene Transcription in Human Brain Cells

doi:

Figure Lengend Snippet: Functional effects of C/EBPβ, C/EBPγ, and NF-IL6 on HIV-1 LTR-driven transcription in human microglial and glial cells. (A) Histogram showing relative CAT activities in microglial and oligodendroglioma TC-620 cells transfected with the LTR-CAT reporter, either alone or in the presence of the indicated expression vectors. The control parental MSV vector contains no cDNA insert. Vectors expressing the mouse C/EBPβ and the human NF-IL6 protein were used. (B) Western blot analysis of nuclear proteins (10 μg) extracted from cells transfected for 48 h with the indicated expression vectors (0.5 pmol). Two concentrations (0.5 and 1.0 pmol) were used for C/EBPβ, as indicated. Blots were probed with the mouse, rat, and human protein-reactive anti-C/EBPβ antibodies (sc-150X; Santa Cruz Biotechnology). Arrows show the endogenous and overexpressed human NF-IL6 protein, the overexpressed mouse C/EBPβ protein, and the endogenous and overexpressed C/EBPγ protein.

Article Snippet: The same gel was cut in two and probed with anti-C/EBPβ (reactive with C/EBPβ and NF-IL6; Santa Cruz Biotechnology) (A) and anti-C/EBPγ (B).

Techniques: Functional Assay, Transfection, Expressing, Plasmid Preparation, Western Blot

Journal:

Article Title: Functional Interactions between C/EBP, Sp1, and COUP-TF Regulate Human Immunodeficiency Virus Type 1 Gene Transcription in Human Brain Cells

doi:

Figure Lengend Snippet: Regulation of HIV-1 gene transcription by NF-IL6, COUP-TF, and Sp1 in human microglial and glial cells. (A) Transient expression experiments were performed by cotransfecting HIV-1 LTR-CAT (1 pmol) with vectors expressing NF-IL6 (0.5 pmol), COUP-TF (0.2 pmol), and Sp1 (0.5 pmol) as indicated. Histograms show CAT activities expressed relative to the value obtained with the LTR-CAT reporter vector. Values correspond to an average of at least three independent experiments done in duplicate. (B) Western blot analysis of nuclear proteins (10 μg) extracted from cells transfected for 48 h with the indicated expression vectors (0.5 pmol). Blots were probed with the mouse, rat, and human protein-reactive anti-C/EBPβ antibodies (sc-150X; Santa Cruz Biotechnology), which recognizes the endogenous and overexpressed human NF-IL6 protein.

Article Snippet: The same gel was cut in two and probed with anti-C/EBPβ (reactive with C/EBPβ and NF-IL6; Santa Cruz Biotechnology) (A) and anti-C/EBPγ (B).

Techniques: Expressing, Plasmid Preparation, Western Blot, Transfection