Journal: The Journal of biological chemistry

Article Title: Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase.

doi: 10.1074/jbc.271.7.3608

Figure Lengend Snippet: FIG. 1. Silver-stained 16.4% (w/v) T (total acrylamide), 1.9% (w/v) C (bisacrylamide) Tricine SDS-polyacrylamide gel show- ing the LPS profiles of E. coli K12 strains. 1, x705; 2, x711; 3, x711(pJB2); 4, Y10; 5, D21e7 (rfa-1); 6, CS2051 (has a deletion elimi- nating rfaG, rfaP, rfaM, rfaN, and rfaB); 7, D31m4 (rfa-229, rfa-230).

Article Snippet: 2.5 mg of cellular RNA from E. coli strain x711 (pJB2), 0.6 mg of pJB2 DNA, and 5 and 10 mg of cellular RNA from E. coli strain x711 were spotted onto a Zeta Probe membrane (Bio-Rad).

Techniques: Staining

Journal: The Journal of biological chemistry

Article Title: Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase.

doi: 10.1074/jbc.271.7.3608

Figure Lengend Snippet: FIG. 2. Physical map of the lpcA region. Vector sequences are not shown. pE4021 is a cosmid clone containing chromosomal DNA from E. coli W3110, including the region from 4.9 to 5.8 min. RNHQ, PEPD, GPTA, PHOE, and PROAB indicate the location of sequenced genes. pJB1 contains a 14-kb EcoRI fragment cloned from pE4021. pJB2 and pJB8 contain a 3-kb BamHI fragment cloned from pJB1 into different vectors. pJB2-9 to pJB2-34 indicate the various deletions of pJB2 span- ning the lpcA region. pJB15 indicates the DNA insert used for construc- tion of DIG-labeled riboprobes. ORF1 and ORF2 are two open reading frames found on opposite strands of the DNA. The direction of tran- scription of lpcA is indicated by the arrow beneath ORF2. The comple- mentation of the novobiocin supersensitivity phenotype by the deletion clones is indicated: R, successful complementation; S, unsuccessful complementation. Restriction enzymes indicated are: A, AvaII; B, BamHI; Bs, BstEII; E, EcoRI; Ev, EcoRV; Hc, HincII; P, PvuI.

Article Snippet: 2.5 mg of cellular RNA from E. coli strain x711 (pJB2), 0.6 mg of pJB2 DNA, and 5 and 10 mg of cellular RNA from E. coli strain x711 were spotted onto a Zeta Probe membrane (Bio-Rad).

Techniques: Plasmid Preparation, Clone Assay, Labeling

Journal: The Journal of biological chemistry

Article Title: Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase.

doi: 10.1074/jbc.271.7.3608

Figure Lengend Snippet: FIG. 4. A schematic representation of the proposed events leading to the chromosomal deletion of the lpcA locus in E. coli strain x711. Panel A, chromosomal map of E. coli K12 strain x705. RNHQ, LPCA, PROAB, IS30A, IS5A, IS1B, and IS30 indicate the location of sequenced genes. Panel B, Southern blot showing chromo- somal DNA profiles of E. coli strains x711 and x705 probed with a 14-kb EcoRI DIG-11-dUTP-labeled DNA probe. M, l HindIII molecular weight markers; 1, x711 DNA digested with EcoRI; 2, x705 DNA di- gested with EcoRI; 3, pJB2 digested with BamHI; 4, pJB1 digested with EcoRI. Panel C, restriction maps of pJB1 and pJB16 showing identical nucleotide sequence (hatched box) and the IS5 element (open box). Panel D, transposition of the IS5A insertion element from approximately 5.9 min to 5.2 min followed by replication of the element, and chromosomal map of E. coli strain x711 showing the resulting deletion of the lpcA locus. Restriction endonucleases indicated are: E, EcoRI.

Article Snippet: 2.5 mg of cellular RNA from E. coli strain x711 (pJB2), 0.6 mg of pJB2 DNA, and 5 and 10 mg of cellular RNA from E. coli strain x711 were spotted onto a Zeta Probe membrane (Bio-Rad).

Techniques: Southern Blot, Labeling, Molecular Weight, Sequencing

Journal: The Journal of biological chemistry

Article Title: Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase.

doi: 10.1074/jbc.271.7.3608

Figure Lengend Snippet: FIG. 5. Reversed-phase high performance liquid chromatogra- phy analyses of carbohydrates synthesized by E. coli strains x711 and x711(pJB2) cell extracts following incubation with 1.0 mmol of sedoheptulose 7-phosphate. Panel A, x711 incubated 60 min. Panel B, x711(pJB2) incubated 2 min. Panel C, x711(pJB2) incu- bated 60 min without sedoheptulose 7-phosphate. Panel D, x711(pJB2) boiled extract incubated 60 min. Large arrow indicates the retention peak of the phosphorylated product. Small arrow indicates the reten- tion peak of sedoheptulose 7-phosphate.

Article Snippet: 2.5 mg of cellular RNA from E. coli strain x711 (pJB2), 0.6 mg of pJB2 DNA, and 5 and 10 mg of cellular RNA from E. coli strain x711 were spotted onto a Zeta Probe membrane (Bio-Rad).

Techniques: Synthesized, Incubation

Journal: The Journal of biological chemistry

Article Title: Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase.

doi: 10.1074/jbc.271.7.3608

Figure Lengend Snippet: FIG. 6. Effect of alkaline phosphatase treatment in the reac- tion products analyzed by reversed-phase high performance liquid chromatography. Upper panel, HPLC profile of x711(pJB2) extract incubated with 1.0 mmol of sedoheptulose 7-phosphate (SED- 7-P) and treated with alkaline phosphatase (4 units) prior to derivat- ization with ABEE. Arrow indicates the location of the reaction peak of the reaction product in the absence of alkaline phosphatase treatment. Lower panel, HPLC profile of authentic glyceromannoheptose derivat- ized with ABEE. ABEE, p-aminobenzoic ethyl ester; AP, alkaline phos- phatase; GMH, glyceromannoheptose.

Article Snippet: 2.5 mg of cellular RNA from E. coli strain x711 (pJB2), 0.6 mg of pJB2 DNA, and 5 and 10 mg of cellular RNA from E. coli strain x711 were spotted onto a Zeta Probe membrane (Bio-Rad).

Techniques: High Performance Liquid Chromatography, Incubation

Journal: Endocrinology

Article Title: Induction of 3beta-hydroxysteroid dehydrogenase/isomerase type 1 expression by interleukin-4 in human normal prostate epithelial cells, immortalized keratinocytes, colon, and cervix cancer cell lines.

doi: 10.1210/endo.140.10.7038

Figure Lengend Snippet: FIG. 8. Stat6 activation by IL-4. Normal human prostate epithelial cells (PrEC) in primary culture, LnCAP and PC-3 prostate cancer cells, ZR-75–1 and BT-20 breast cancer cells, HaCaT human immor- talized keratinocytes, HT-29 and Caco-2 human colon cancer cells, ME-180 human cervix cancer cells or JAR and JEG-3 human cho- riocarcinoma cells were incubated in the presence of absence of IL-4 (10 ng/ml for 30 min). Analysis of Stat6 activation using EMSA was performed as described in Materials and Methods using a well es- tablished Stat6 responsive element derived from the IgE-promoter. A Stat6 antibody was included in the binding reaction where indicated.

Article Snippet: The membrane was probed with a Stat6 antibody (Santa Cruz Biotechnology, Inc., Santa Clara, CA, catalog no. SC-621X).

Techniques: Activation Assay, Incubation, Derivative Assay, Binding Assay

Journal: Endocrinology

Article Title: Induction of 3beta-hydroxysteroid dehydrogenase/isomerase type 1 expression by interleukin-4 in human normal prostate epithelial cells, immortalized keratinocytes, colon, and cervix cancer cell lines.

doi: 10.1210/endo.140.10.7038

Figure Lengend Snippet: FIG. 7. Stat6 expression in cell types derived from peripheral tissues. Ten micrograms of total cell extract from ZR-75–1 and BT-20 breast cancer cells, normal human prostate epithelial cells (PrEC) in pri- mary culture, LnCAP and PC-3 prostate cancer cells, HaCaT human immortalized keratinocytes; HT-29and Caco-2 human colon cancer cells, ME-180 human cervix cancer cells, JAR and JEG-3 human choriocarcinoma and 293 human embryonic kidney cells were sepa- rated on SDS-PAGE. Western blot analysis was performed as de- scribed in Materials and Methods. Equal sample loading and transfer efficiency was confirmed by staining of the membrane with Ponceau Red.

Article Snippet: The membrane was probed with a Stat6 antibody (Santa Cruz Biotechnology, Inc., Santa Clara, CA, catalog no. SC-621X).

Techniques: Expressing, Derivative Assay, SDS Page, Western Blot, Staining, Membrane

Journal: Endocrinology

Article Title: Induction of 3beta-hydroxysteroid dehydrogenase/isomerase type 1 expression by interleukin-4 in human normal prostate epithelial cells, immortalized keratinocytes, colon, and cervix cancer cell lines.

doi: 10.1210/endo.140.10.7038

Figure Lengend Snippet: FIG. 9. IL-4-activated Stat6 binds to consensus Stat6 sequence sites in the 3b-HSD type 1 gene promoter. Normal human prostate epi- thelial cells (PrEC) in primary culture were incubated in the presence of absence of IL-4 (10 ng/ml for 30 min). Analysis of Stat6 activation using EMSA was performed as described in Materials and Methods using the 3b-HSD type 1 Stat6#1 and Stat6#2 probes from the 3b- HSD type gene promoter. A Stat6 antibody was included in the bind- ing reaction where indicated.

Article Snippet: The membrane was probed with a Stat6 antibody (Santa Cruz Biotechnology, Inc., Santa Clara, CA, catalog no. SC-621X).

Techniques: Sequencing, Incubation, Activation Assay

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Dose-dependent neuroprotection of VEGF₁₆₅ in Huntington's disease striatum.

doi: 10.1038/mt.2013.132

Figure Lengend Snippet: Figure 2 Neuroprotective and anti-aggregating effects of vascular endothelial growth factor 165 (VEGF165) in an in vitro SH-SY5Y model of Huntington’s disease. (a) SH-SY5Y neuroblastoma cells were transduced with lentiviral vector expressing wild-type Htt (18Q), Exp-Htt (82Q) alone or in combination with lentiviral vector encoding human VEGF165 (82Q + VEGF) or control lentiviral vector (82Q + ΔLNGFR). Ninety-six hours after transduction, cell death was assessed by trypan blue staining. Expression of VEGF with 82Q showed a significant reduction in trypan blue positive cells compared with 82Q alone (n = 4, mean ± SEM, *P < 0.05, Student’s t-test). Equivalent levels of trypan blue positive cells were observed in the non-transduced and 18Q-transduced groups. (b) The experiment was repeated and cell apoptosis assessed by Hoechst dye staining. Bright staining indicated cells undergoing apoptosis (examples of apoptotic cells circled). Coexpression of VEGF with 82Q showed a significant reduction in Hoechst positive cells compared with 82Q alone (n = 4, mean ± SEM, ***P < 0.001, Student’s t-test). (c) SH-SY5Y cells were transduced with lentiviral vector encoding wild-type Htt (18Q), Exp-Htt (82Q) alone or in combination with lentiviral vector encoding human VEGF165 (82Q + VEGF), negative control lentiviral vector (82Q + ΔLNGFR) or positive control lentiviral vector encoding Hsp70 (82Q + Hsp70). Ninety-six hours after transduction, the forma- tion of huntingtin inclusions was assessed by EM48 antibody and DAB staining. Quantitative analysis demonstrates that coexpression of VEGF with 82Q significantly reduced the number of huntingtin aggregates compared with 82Q alone (n = 4, mean ± SEM, *P < 0.05, Student’s t-test). Scale bars represent 50 μm.

Article Snippet: Separated proteins were transferred onto nitrocellulose membrane (Hybond-ECL, GE Healthcare, Chalfont St Giles, UK), blocked with PBS/ Tween-20 (0.1% (v/v)) with 5% (w/v) milk powder) and probed with antibodies to VEGF165 (Santa Cruz, Santa Cruz, CA; sc-7269) or HSP70 (Santa Cruz; sc-59569), c-Myc (tag HTT) (Santa Cruz; sc-56634) and GAPDH (Santa Cruz; sc-47724).

Techniques: In Vitro, Transduction, Plasmid Preparation, Expressing, Control, Staining, Negative Control, Positive Control

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Dose-dependent neuroprotection of VEGF₁₆₅ in Huntington's disease striatum.

doi: 10.1038/mt.2013.132

Figure Lengend Snippet: Figure 3 Neuroprotective effects of vascular endothelial growth factor 165 (VEGF165) in an in vitro primary striatal neuronal culture model of Huntington’s disease. Primary striatal neuronal cultures were transduced with lentiviral vectors encoding ΔLNGFR or 18Q as controls and 82Q alone or in combination with ΔLNGFR, Hsp70 or VEGF165. Six weeks after transduction striatal cultures were fixed and stained for hun- tingtin aggregates (EM48 antibody) and NeuN followed by DAB stain- ing. Huntingtin aggregates were observed in all groups expressing 82Q, but a reduction of aggregates was observed in cells cotransduced with 82Q and the positive control Hsp70. Neuronal survival was assessed with NeuN staining. Striatal neuronal cultures coinfected with 82Q and VEGF showed a significant enhancement of cell survival compared with 82Q alone (n = 3, mean ± SEM, ***P < 0.001, Student’s t-test) and 82Q plus Hsp70 or ΔLNGFR control (n = 3, mean ± SEM, **P < 0.01, one way analysis of variance). Scale bar represents 50 μm.

Article Snippet: Separated proteins were transferred onto nitrocellulose membrane (Hybond-ECL, GE Healthcare, Chalfont St Giles, UK), blocked with PBS/ Tween-20 (0.1% (v/v)) with 5% (w/v) milk powder) and probed with antibodies to VEGF165 (Santa Cruz, Santa Cruz, CA; sc-7269) or HSP70 (Santa Cruz; sc-59569), c-Myc (tag HTT) (Santa Cruz; sc-56634) and GAPDH (Santa Cruz; sc-47724).

Techniques: In Vitro, Transduction, Staining, Expressing, Positive Control, Control

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Dose-dependent neuroprotection of VEGF₁₆₅ in Huntington's disease striatum.

doi: 10.1038/mt.2013.132

Figure Lengend Snippet: Figure 5 Neuroprotective and anti-aggregating effects of low dose vascular endothelial growth factor 165 (VEGF165) in an in vivo model of HD. (a) Immunocytochemical detection of DARPP-32 (top panels) and NeuN (bottom panels) was performed with specific antibodies (see Materials and Methods). 82Q-induced neurodegeneration confirmed by DARPP-32 and NeuN depletion is attenuated by VEGF expression. Graphical represen- tation of the total volume depletion for DARPP-32 and NeuN for 82Q + ΔLNGFR control and 82Q + VEGF–infected striatums (n = 4, mean ± SEM, *P < 0.05, **P < 0.001, Student’s paired t-test). (b) The number of striatal 82Q-induced Htt aggregates was significantly reduced by coexpression of VEGF (n = 4, mean ± SEM, *P < 0.05, Student’s paired t-test, 82Q + ΔLNGFR control versus 82Q + VEGF infection). (c) Reactive astrocytes were assessed by GFAP staining (82Q + ΔLNGFR control versus 82Q + VEGF infection). Reactive astrocytes were observed in both groups around the sites of injection in the cortex, corpus collosum and the striatum (arrow heads). (d) Striatal 82Q vector spread as determined by GFP reporter expression is equivalent for both 82Q + ΔLNGFR control and 82Q + VEGF–transduced groups (n = 4, mean ± SEM). (e) Percentage weight increase of animals following 82Q + VEGF or 82Q + ΔLNGFR injection. Scale bars represent 200 μm.

Article Snippet: Separated proteins were transferred onto nitrocellulose membrane (Hybond-ECL, GE Healthcare, Chalfont St Giles, UK), blocked with PBS/ Tween-20 (0.1% (v/v)) with 5% (w/v) milk powder) and probed with antibodies to VEGF165 (Santa Cruz, Santa Cruz, CA; sc-7269) or HSP70 (Santa Cruz; sc-59569), c-Myc (tag HTT) (Santa Cruz; sc-56634) and GAPDH (Santa Cruz; sc-47724).

Techniques: In Vivo, Expressing, Control, Infection, Staining, Injection, Plasmid Preparation

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Dose-dependent neuroprotection of VEGF₁₆₅ in Huntington's disease striatum.

doi: 10.1038/mt.2013.132

Figure Lengend Snippet: Figure 7 Extensive neuroinflammation with high dose vascular endothelial growth factor 165 (VEGF165) in an in vivo model of HD. (a) 82Q + high dose VEGF–injected brains: widespread macrophages/ monocyte (ED1, green staining) and CD4+ T cell (CD4, red staining) infiltration was observed in the ipsilateral side in regions such as the (ii, iii) striatum (str), (vi, vii) corpus collosum (cc), (x, xi) forceps major (fm), and (xiv,xv) piriform cortex (pc). (iv) Some cytotoxic T cells were also observed in the striatum close to the site of injection. No inflammatory markers were detected on the contralateral side (i,v,ix,xiii). (b) 82Q + low dose VEGF–injected brains: No macrophage/monocyte infiltration (ED1, green staining) or CD4+ T cells (CD4, red staining) were observed in the striatum (str) or corpus collosum (cc) of rats injected with either 82Q + ΔLNGFR or 82Q + low dose VEGF (i–viii). Scale bar represents 100 μm.

Article Snippet: Separated proteins were transferred onto nitrocellulose membrane (Hybond-ECL, GE Healthcare, Chalfont St Giles, UK), blocked with PBS/ Tween-20 (0.1% (v/v)) with 5% (w/v) milk powder) and probed with antibodies to VEGF165 (Santa Cruz, Santa Cruz, CA; sc-7269) or HSP70 (Santa Cruz; sc-59569), c-Myc (tag HTT) (Santa Cruz; sc-56634) and GAPDH (Santa Cruz; sc-47724).

Techniques: In Vivo, Injection, Staining

Journal: BMC Cancer

Article Title: Increased expression of system large amino acid transporter (LAT)-1 mRNA is associated with invasive potential and unfavorable prognosis of human clear cell renal cell carcinoma

doi: 10.1186/1471-2407-13-509

Figure Lengend Snippet: Expression of phosphorylated S6 ribosomal protein (Ser-235/236) (32 kDa) and beta actin (42 kDa) proteins in the primary tumor tissues using Western blotting. M; marker. N; non-tumor tissue. T; primary tumor tissue with metastatic lesions. Each number corresponds to a case number.

Article Snippet: After the membrane was blocked, the bound proteins were probed with an anti-phosphorylated S6 ribosomal protein (Ser-235/236) antibody, 2 F9, which is an anti-human primary antibody and was raised in rabbits (Cell Signaling Technology, Inc; # 4856), and a primary antibody for β-actin (Millipore; # 1501R Bedford, MA).

Techniques: Expressing, Western Blot, Marker

Journal: BMC Cancer

Article Title: Increased expression of system large amino acid transporter (LAT)-1 mRNA is associated with invasive potential and unfavorable prognosis of human clear cell renal cell carcinoma

doi: 10.1186/1471-2407-13-509

Figure Lengend Snippet: Expression of phosphorylated S6 ribosomal protein (Ser-235/236). a ; tumor. b ; grade. c ; pT stage. d ; microscopic vascular invasion. e ; metastasis. The data show the 95% confidential interval.

Article Snippet: After the membrane was blocked, the bound proteins were probed with an anti-phosphorylated S6 ribosomal protein (Ser-235/236) antibody, 2 F9, which is an anti-human primary antibody and was raised in rabbits (Cell Signaling Technology, Inc; # 4856), and a primary antibody for β-actin (Millipore; # 1501R Bedford, MA).

Techniques: Expressing

Journal: BMC Cancer

Article Title: Increased expression of system large amino acid transporter (LAT)-1 mRNA is associated with invasive potential and unfavorable prognosis of human clear cell renal cell carcinoma

doi: 10.1186/1471-2407-13-509

Figure Lengend Snippet: Spearman rank correlation coefficient relationship. X axis is an independent variable. Y axis is a dependent variable. LAT1 mRNA levels positively correlated with phosphorylated S6 ribosomal protein (Ser-235/236) levels in primary tumor tissues.

Article Snippet: After the membrane was blocked, the bound proteins were probed with an anti-phosphorylated S6 ribosomal protein (Ser-235/236) antibody, 2 F9, which is an anti-human primary antibody and was raised in rabbits (Cell Signaling Technology, Inc; # 4856), and a primary antibody for β-actin (Millipore; # 1501R Bedford, MA).

Techniques: