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Production of LTs from HCMV-infected HPASMCs. HPASMCs were infected with HCMV at an MOI of 10 for 1, 3, 7, and 10 d, collected, washed, and incubated with 2 mM Ca2+ and 2.5 μM ionophore for 5 min at 37°C. LTB4 and cys-LTs were purified by solid-phase extraction and HPLC and measured with an enzyme immunoassay. n = 6. Values are mean ± SD. **, P < 0.01 versus uninfected cells. Pretreatment with bFGF inhibits Fas-mediated apoptosis. (A) FH2 cells were pretreated with 100 ng/ml EGF, 100 ng/ml IGF or 10 ng/ml bFGF for 16 h, and then stimulated with 200 ng/ml agonistic anti-Fas mAb RK-8 for the indicated times. Cell viability was measured by amido black staining assay (see Materials and Methods). (B) Activation of MAPK in FH2 cells treated with EGF, IGF, or bFGF was detected by Western blotting with anti–phospho-MAPK antibody. Expression level of MAPK was confirmed by Western blotting with anti-MAPK antibody. (C and D) FH2 cells were pretreated with or without 10 ng/ml bFGF for 16 h and stimulated with anti-Fas mAb RK-8 for the indicated times. Activation of caspases in cell lysate was measured using the fluorescent substrate IETD-MCA and DEVD-MCA for caspase-8/6 (C) and caspase-3/7 (D), respectively. c-MYC induction confers resistance to PI3K/mTOR inhibition(a) Western blot analysis of ICN1 or control MCF10A cells treated with BEZ-235 (pg/μl) as indicated for 24 hours. Total lysates were probed with an antibody against phosphorylated ribosomal S6 kinase (Thr371) and total mTOR as a loading control (see Supplementary Fig. 20 for an uncropped version). (b) Data from the screen shows c-MYC as a significant hit for resistance to BEZ-235. (c) Relative c-MYC mRNA levels in ICN1 and c-MYC cells as determined by qRT-PCR. Shown is the fold change compared to wild-type MCF10A cells and standard deviations of 3 replicates. (d) Dose-response curve of c-MYC or control MCF10A cells treated with BEZ-235. Cells were treated for 5 days as indicated and relative cell number was measured. The data represent four independent experiments were performed in triplicate and error bars indicate standard deviations. (e) Quantitative RT-PCR of c-MYC expression in wild-type MCF10A or ICN1 cells transfected with Luciferase siRNA and ICN1 cells transfected with c-MYC siRNA pool (ICN1+si). Standard deviations of 3 replicates are indicated. (f) Dose-response curve of cells in (e) treated with BEZ-235 or vehicle for 5 days. Three replicates were performed; error bars indicate standard deviation. (g) Oncomine analysis (see Methods) of c-MYC gene copy number in PI3K/mTOR inhibitor sensitive or resistant cell lines. The red-boxed area indicates cell lines with c-MYC gene amplification (Chi square P value is indicated). Multi-locus sequence typing (MLST) of multi-drug resistant (MDR) serotype 19A isolates (n = 97) obtained from the Canadian Bacterial Diseases Surveillance Network during pre-PCV 7 introduction, vaccine introduction, and post-vaccine introduction eras. Sequence types (ST) are depicted as a percent of all MDR 19 isolates for that period. ST320 has emerged as the singular dominant sequence type amongst MDR 19A isolates in the post-PCV7 era. Novel implies a collection of strains for which no sequence type (ST) was identified within the MLST http://www.mlst.net/ database at this time but submissions have been made and are summarized in Additional file 2. eBURST summary data for MDR 19A, MDR 19F (n = 30) and susceptible 19A (n = 19) controls are appended in Additional file 1http://www.pillailab.com/suppdata/index.html. Map of the Vk167/PEPS transgene and EPS analysis. (A) Vk167/PEPS transgene and sequence of the EPS insert. The restriction sites within the EPS are indicated. (B) An example of PAGE from an EPS analysis. PCR products from nine clones were digested with EcoRV (E) or PvuII (P) and resolved in 18% acrylamide gel. Unmutated clones (12G, 2E, 2B, and 2D) show two large bands flanking the EPS region and ladders of small bands. In case of EcoRV digestion, the small bands are 20, 18, 16, 14, 12, and 10 bp (12- and 10-bp bands are invisible here). In case of PvuII digestion, the small bands are 19, 17, 15, 13, and 11 bp (11-bp bands are invisible). An asterisk indicates mutated clones. Clone 8G, for example, has a mutation in the fourth PvuII site (PD), so the 15- and 17-bp bands are gone and a 32-bp band has appeared.
Overall (OS) (lower) and Cancer Specific (CSS) (upper) actuarial survival curves. CpG ODN enhance HEL-specific IFN-γ production by BALB/c splenocytes. Mice were immunized as in Fig. 1 with 100 μg ODN/mouse in A and 30 μg ODN/mouse in panel B. After 3 wk, splenocytes were isolated and incubated with HEL (closed circles) or medium alone (open circles). ELISA spot assay was performed and spots were quantitated by a computerized image analysis program. Each point represents the mean number of spots per well for one mouse (assayed in duplicate); horizontal bars indicate the mean of points for each group of mice. Similar results were observed in five independent experiments with CpG- and non–CpG ODN in BALB/c mice. Effect of MIP-3α on intracellular free calcium in DCCR2 receptor–expressing HEK 293 cells. HEK 293 cells transiently expressing the DCCR2 receptor were loaded with Fluo-3AM and exposed to either vehicle (buffer) or to 50 nM–1 μM concentrations of MIP-3α. Calcium fluorometry was conducted as described in Materials and Methods. Tracings represent the means of eight experiments conducted in single determinations. GANT61- and Cyclopamine- induced cell death Percentage of survival among C57BL/6 B-cell (μMT), CD4 T-cell (CD4-/-) and CD8 T-cell (CD8-/-)-deficient, HK-vaccinated mice. Two weeks post vaccination, mice were challenged with 2 × 107 CFU/100 μl of live B. mallei by intraperitoneal injection. B-cell-deficient mice demonstrated a 50% decreased survival (p = 0.0888) compared to that of the wild-type mice with a MST of 35.5 days (n = 6). CD4-/- and CD8-/- mice resulted in 60% (p = 0.1343) and 0% reduced survival, respectively (n = 5).
Schematic experimental setup used for nonlinear imaging. The red path corresponds to the fundamental excitation beam centered at 965nm, the blue path to the SHG emission and the green path to the TPEF emission. L# are lenses; GM are the galvanometric mirrors; OL is the objective lens (40x, NA = 1.3), CO is the condenser optics (NA = 1.4); F1 ad F2 are the band pass filters (F1 transmittance = 330 – 670 nm and F2 transmittance = 475 – 485 nm); and PMT are the photo multiplier tubes. The effects of MTSES and DTT on the transient currents by D444C. Currents recorded in the presence of 2 mM L-aspartate were subtracted from currents recorded in its absence in oocytes expressing either D444C-EAAC1 (A–C) or D444S-EAAC1 (D–F). The transient currents were measured before (A and D) or after the application of 2 mM MTSES (B and E) and after MTSES and 50 mM DTT (C and F). Typical oocytes are shown (n = 3). Overview of the distance methods and the tests applied. The picture describes the pure-distance framework developed in the course of the present study for testing methods of deriving host character data and/or distance matrices from associate character data. The location of the methods for character-character transformation (ENT, FRQ, CON, MOD) and for distance-distance (MIN, PBC) transformation is indicated. Integrating the SIZ distances (that are biologically meaningless in the case of cloned sequences) allows us to test for a potential sampling size bias. Abbreviations: CM, character matrix; DM, distance matrix. See the methods section for the abbreviations for the transformation methods. Hexamerization assembles a catalytically robust ATPase complex. (a) ATPase activity as a function of E1HD protein concentration. Reactions were assembled with (solid line) and without oligonucleotide (dashed line) and Pi released determined after 3 min at 22°C. (b) Oligonucleotide stimulates E1HD hexamerization in the presence of ATP. At 17.4 µM ∼40% of the E1HD protein is in the oligomeric form (left) and oligonucleotide stimulates oligomerization (right). Stimulation index of IP-10 and IFN-γ in active TB, LTBI and a control group.Horizontal lines indicate medians. Controls are represented by ○, active TB by ▵, and LTBI by □. Open symbols represent IP-10, closed symbols represent IFNγ samples.
Determination of β-gal expression in R/gal and RS/gal transfectants. Serial dilutions of NP-40 lysates of R/gal (open triangles) and RS/gal (closed triangles) transfectants or untransfected RMA cells (closed circles) were incubated with the chromogenic substrate X-gal and enzyme activity was determined by measuring the absorbance at 620 nm. Association between T1 mapping and scar tissue analyses using LGE. (a) Uptake of folates over the erythrocytic cycle of P. falciparum. Synchronised HB3 cultures (initial 4% parasitaemia) were sampled every 7–9 h, cells spun down, washed with pABA/folate depleted medium and incubated at 37 °C in 1 ml RPMI1640 containing 10 ng/ml each of pABA and tritium-labelled folinic acid (squares) or 5-MeTHF (circles) before extraction and counting of labelled folate from saponin-freed parasites (left vertical axis). The number of infected erythrocytes per unit volume present at each time point is also shown (triangles; right vertical axis). (b) Temperature dependence of folinic acid transport into P. falciparum. Saponin-freed parasites were washed with culture medium devoid of folate/pABA, then incubated in PBS, 20 mM glucose, 38 nM 3H-folinic acid. Parasites and reagents were prewarmed separately to the desired temperature in a thermal gradient block, mixed to initiate uptake and incubated for 1 h, before harvesting the parasites and counting imported label. Effect of silica particles on membrane damage. Cellular membrane damage in HaCaT cells after incubation with nSP70 (circles), nSP300 (squares) and mSP1000 (diamonds) for 24 h was evaluated by the LDH release assay. The percentage cellular membrane damage was calculated relative to the negative (medium) controls. Data are presented as means ± SD (n = 3).*P < 0.01 vs same dose of nSP300 and mSP1000. Survival of BALB/c mice immunised with recombinant Omp3 or Omp7 and challenged with B. pseudomallei.Mice were immunised with either Omp3 (open triangles) or Omp7 (open squares) prior to challenge with 1×106 cfu B. pseudomallei by the i.p. route of infection. Both groups immunised with either Omp3 or Omp7 displayed 50% survival rate up to 21 days post-infection. All the control mice (closed squares) received E. coli BL21 (DE3) cells in FIA followed by challenge, and died by day 9 post-challenge.
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