Journal: International Journal of Molecular Sciences

Article Title: Anti-Metastatic Effect of Pyruvate Dehydrogenase Kinase 4 Inhibition in Bladder Cancer via the ERK, SRC, and JNK Pathways

doi: 10.3390/ijms232113240

Figure Lengend Snippet: Expression of PDK4 in siRNA-treated bladder cancer cell lines. ( A ) Fold change of PDK4 mRNA expression in T24 and J82 cells. ( B ) Protein expression of PDK4 in T24 and J82 cells. All data represent means ± SD of three independent experiments (* p < 0.05, ** p < 0.01 NC vs. T). M: mock, N: negative control, T: PDK4 siRNA treated.

Article Snippet: Anti-PDK4 antibody was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Expressing, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Anti-Metastatic Effect of Pyruvate Dehydrogenase Kinase 4 Inhibition in Bladder Cancer via the ERK, SRC, and JNK Pathways

doi: 10.3390/ijms232113240

Figure Lengend Snippet: Migration and invasion assay of PDK4 knockdown bladder cancer cells. ( A ) Migration assay. ( B ) Invasion assay. All data represent means ± SD of three independent experiments (* p < 0.05, ** p < 0.01 NC vs. T). M: mock, N: negative control, T: PDK4 siRNA treated.

Article Snippet: Anti-PDK4 antibody was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Migration, Invasion Assay, Knockdown, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Anti-Metastatic Effect of Pyruvate Dehydrogenase Kinase 4 Inhibition in Bladder Cancer via the ERK, SRC, and JNK Pathways

doi: 10.3390/ijms232113240

Figure Lengend Snippet: PDK4-related protein expression in PDK4 knockdown bladder cancer cells. β-actin is used as an internal control. M: mock, N: negative control, T: PDK4 siRNA treated. All data represent means ± SD of three independent experiments (* p < 0.05, ** p < 0.01 NC vs. T). M: mock, N: negative control, T: PDK4 siRNA treated.

Article Snippet: Anti-PDK4 antibody was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Expressing, Knockdown, Control, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Anti-Metastatic Effect of Pyruvate Dehydrogenase Kinase 4 Inhibition in Bladder Cancer via the ERK, SRC, and JNK Pathways

doi: 10.3390/ijms232113240

Figure Lengend Snippet: Xenograft model of PDK4 knockdown J82 cells. ( A ) Tumor growth. (* p < 0.05, ** p < 0.01 Day 0 vs. Each day). Ctrl: wild type, KD: knockdown. ( B ) Gross appearance. ( C ) Representative image of immunohisto-chemistry. (* p < 0.05, ** p < 0.01 Ctrl vs. KD).

Article Snippet: Anti-PDK4 antibody was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Knockdown, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Anti-Metastatic Effect of Pyruvate Dehydrogenase Kinase 4 Inhibition in Bladder Cancer via the ERK, SRC, and JNK Pathways

doi: 10.3390/ijms232113240

Figure Lengend Snippet: PDK4 expression in human bladder cancer specimen. ( A ) Relative PDK4 mRNA expression in normal and bladder cancer specimen. ( B ) Representative image of immunohisto-chemistry of PDK4 protein. All data represent means ± SD of three independent experiments (** p < 0.01 normal vs. T stages).

Article Snippet: Anti-PDK4 antibody was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Expressing, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Anti-Metastatic Effect of Pyruvate Dehydrogenase Kinase 4 Inhibition in Bladder Cancer via the ERK, SRC, and JNK Pathways

doi: 10.3390/ijms232113240

Figure Lengend Snippet: MS-based quantitative proteomic profiling of phosphorylation in bladder cancer cell lines. ( A ) Experimental design of proteomic analysis. J82 and J82 KD cells were lysed, subjected to in-solution digestion and 18 O labeling, and then mixed at equal protein amounts. Phosphorylated peptides were enriched by TiO 2 . Eluted peptides were analysis using LC-MS/MS. Mass spectrum data were searched in the MaxQuant (version 1.5) database. ( B ) Venn diagrams showing overlap between J82 knock down and J82 control. ( C ) Volcano plot for 209 differentially phosphorylated protein (DRPs) between PDK4 knockdown and controls.

Article Snippet: Anti-PDK4 antibody was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Phospho-proteomics, Labeling, Liquid Chromatography with Mass Spectroscopy, Knockdown, Control

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: The Egr-1 gene is rapidly induced after TCR-mediated activation of the DPK double positive cell line. ( A ) Total RNA isolated from DPK cells cultured with immobilized anti-CD3ε mAb for the indicated times (shown in hours), was subjected to RT-PCR analysis using Egr-1 or CD4 primers. ( B ) Electrophoretic mobility shift assay using nuclear lysates prepared from DPK cells 8 h after activation by immobilized antiCD3ε mAb. Probes contained a single Egr-1 binding site ( left ) or overlapping Egr-1 and Sp1 sites ( right ). ( C ) DPK cells were cultured with DCEK-ICAM fibroblast antigen presenting cells and 1 μm pigeon cytochrome c peptide for the indicated times. Total RNA was isolated and subjected to a competitive RT-PCR assay (see Materials and Methods). Note the different scales for Egr-1 and CD4 mRNA expression.

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Activation Assay, Isolation, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Electrophoretic Mobility Shift Assay, Binding Assay, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: DPK cell differentiation and Egr-2,3 mRNA induction is cyclosporin A sensitive, while Egr-1 mRNA induction is cyclosporin A resistant. ( A , B ) DPK cells were cultured with DCEK-ICAM fibroblast antigen presenting cells and 2 μM pigeon cytochrome c peptide in the presence or absence of 100 ng/ml cyclosporin A, or appropriate dilution of solvent (DMSO) as indicated. Cells were harvested and stained for CD69 after 1 or 3 d in culture ( A ) or stained for CD4 and CD8 after 3 d in culture ( B ). ( C ) RT-PCR analysis of total RNA derived from DPK cells activated for 6 h with immobilized anti-CD3ε mAb in the presence or absence of 300 ng/ml cyclosporin A, using Egr-1, Egr-2 (Krox-20), CD4, CD69 or Egr-3 primers. (*) Also shown for the indicated samples is the relative level of Egr-1 cDNA normalized to expression of CD4 cDNA as determined by competitive RT-PCR assay. The identity of the lower major band in Egr-3 RT-PCR was verified by sequencing.

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Cell Differentiation, Cell Culture, Staining, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Expressing, Sequencing

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: Expression of Egr gene family is dependent upon ras signaling pathways. ( A ) Competitive RT-PCR was used to compare expression of Egr-1 and CD4 genes in DPK or 17N4 cells activated for 6 h with immobilized anti-CD3ε mAb. ( B ) RT-PCR analysis of total RNA derived from DPK or 17N4 cells activated for 6 h with immobilized anti-CD3ε mAb. Independent PCR reactions using Egr-2 (Krox-20), CD4, or Egr-3 primers were performed.

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: Egr-1 mRNA and DNA binding activity in the thymus is MHC dependent. ( A ) Thymocytes derived from wild-type or MHCdeficient mice were two color-stained for CD4 and CD8. ( B ) Competitive RT-PCR was used to determine the level of expression of CD4 and Egr-1 genes in thymocytes derived from wild-type or MHC-deficient mice. ( C ) Electrophoretic mobility shift assay using nuclear lysates prepared from freshly isolated thymocytes derived from wild-type or MHCdeficient mice using a probe containing an Egr-1 binding site. Nuclear extracts derived from 5 × 10 5 cells containing equivalent amounts of protein were used in binding reactions. In some instances as indicated, binding reactions contained anti-Egr-1 antibody or normal rabbit serum (NRS). For comparison, a binding reaction containing recombinant Egr-1 is shown.

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Binding Assay, Activity Assay, Derivative Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Electrophoretic Mobility Shift Assay, Isolation, Recombinant

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: Expression of Egr-1 mRNA in double positive thymocytes. ( A ) Total thymocytes and CD4 + 8 + thymocytes (isolated by cell sorting, 95% DP) derived from the same animal, were assayed for expression of Egr and CD4 mRNA by competitive RT-PCR. Shown is the relative level of Egr-1 cDNA in the sample, normalized to the level of CD4 cDNA. ( B ) Total thymocytes derived from an MHC-deficient mouse were cultured with hamster immunoglobulin-coated or anti-CD3ε mAbcoated beads for 90 min before determination of Egr-1 and CD4 gene expression as in Fig. C .

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Expressing, Isolation, FACS, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: Expression of Egr-1 protein in the thymus. Thin sections of normal thymus ( A–C ) or MHC knockout thymus ( D ) were fixed in formaldehyde and stained with a specific rabbit anti-Egr-1 peptide antiserum ( A , C , D ) or the same antibody preincubated with specific peptide ( B ). Regions of cortex ( C ) and medulla ( M ) are indicated. Sections were counterstained with hematoxylin and photographed at ×20 ( A , B ) or ×40 ( C , D ). C shows a magnification of the same section photographed in A .

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Expressing, Knock-Out, Staining

Journal: The Journal of Experimental Medicine

Article Title: Induction of the Early Growth Response (Egr) Family of Transcription Factors during Thymic Selection

doi:

Figure Lengend Snippet: Expression of Egr-1 protein in thymocyte subsets. Thymocytes from a young adult mouse were 4-color stained for expression of CD4, CD8, Egr-1, and CD69 or CD3, and analyzed by FACS ® as described in Materials and Methods. Indicated in the dot plots are the percentage of thymocytes within each quadrant, or in parenthesis ( upper right dot plot ), the percentage of Egr-1 + thymocytes within each thymocyte subset. Where indicated, staining is shown for gated populations of thymocytes (either Egr-1 + thymocytes as shown in histogram, or CD4 + 8 + thymocytes). Similar results were obtained from three other individual animals.

Article Snippet: Sections were stained with a specific affinity-purified rabbit anti-Egr-1 peptide antiserum ( Santa Cruz Biotechnology ) for 1 h followed by peroxidase-coupled F(ab′) 2 donkey anti–rabbit IgG antiserum (Jackson ImmunoResearch Labs.) for 30 min. For control staining, the anti-Egr-1 peptide antiserum was preincubated for 2 h at room temperature with a 10-fold excess by weight of the immunizing peptide ( Santa Cruz Biotechnology ).

Techniques: Expressing, Staining

Journal: Molecular pharmaceutics

Article Title: Species Differences in the Pharmacology and Toxicology of PEGylated Helper-Dependent Adenovirus

doi: 10.1021/mp100216h

Figure Lengend Snippet: PEGylation alters the expression and function of hepatic cytochrome P450 3A (CYP3A) in the baboon. Full blood chemistry panels were run on baboons given either unmodified or PEGylated helper-dependent adenovirus expressing beta-galactosidase at a dose of 5 × 1011 or 3 × 1012 vp/kg. Changes in serum aspartate aminotransaminase (AST) (A) and serum lactate dehydrogenase (LDH) (B) profiles were noted. All other parameters measured (see Experimental Section under Biochemical and Hematological Analysis of Blood) fell within normal limits for each primate (data not shown). (C) Immunoblot analysis of hepatic CYP3A in male baboons 96 h after a single systemic dose of 5 × 1011 or 3 × 1012 vp/kg of either unmodified or PEGylated helper-dependent adenovirus expressing beta-galactosidase. Protein levels are reported as arbitrary units of relative density with respect to a known protein standard. (D) In vitro catalytic activity of CYP3A microsomal proteins 96 h after vector administration as measured by the production of the isoform-specific metabolite, 6β-hydroxytestosterone. Data represent values obtained from one primate per experimental condition.

Article Snippet: 47 Detection of CYP3A protein was achieved using a rabbit polyclonal antipeptide against human CYP3A4 antibody (1:4000, A4100, Xenotech) and a corresponding horseradish peroxidase conjugated secondary antibody (Cell Signaling Technology, Denver, MA).

Techniques: Expressing, Western Blot, In Vitro, Activity Assay, Plasmid Preparation