Journal: bioRxiv

Article Title: Spatio-temporal requirements of Aurora kinase A in mouse oocytes meiotic spindle building

doi: 10.1101/2024.04.01.587547

Figure Lengend Snippet: (A) Experimental design of WT oocytes treated with MLN during oocyte maturation. Red arrows indicate MLN addition time; blue arrows indicate fixation time. (B, F, J) Schematic of spindles at indicated stages, and the processes analyzed. (C, G, K) Representative confocal images of oocytes fixed at the indicated stages with and without MLN, immuno-stained with PCNT (gray), TACC3 (magenta), Tubulin (green) and DAPI (blue). Scale bars 10 µm. (D, H, L) Quantification of aMTOC parameters in (A), (G), (K) respectively. (E, I, M) Quantification of spindle and LISD parameters in (A), (G), (K) respectively. Black dots: DMSO-treated; gray dots: MLN-treated. See also Figure S1 which contains statistics.

Article Snippet: Primary antibodies and concentrations were used as follows: Pericentrin (Pcnt) (mouse, 1:100; BD Biosciences, #611814); TACC3 (Rabbit, 1:100; Novus Biologicals # NBP2-67671), Alpha Tubulin polyclonal (Sheep, 1:100; Cytoskeleton #ATN02); phosphorylated ABC (rabbit, 1:100; Cell Signaling Technology, #2914), phosphorylated CDC25B (1:100; Signalway Antibodies #11949); phosphorylated INCENP (Gift from Michael Lampson), Human anti-ACA (1:30, Antibodies Incorporated #15-234).

Techniques: Staining

Journal: bioRxiv

Article Title: Spatio-temporal requirements of Aurora kinase A in mouse oocytes meiotic spindle building

doi: 10.1101/2024.04.01.587547

Figure Lengend Snippet: (A) Experimental design of BC-KO oocytes treated with MLN; Red arrows indicate MLN addition time; blue arrows indicate fixation time. (B, F, J) Schematic of spindles at the indicated stages, and the processes analyzed. (C, G, K) Representative confocal images of oocytes fixed at the indicated stages with and without MLN, immuno-stained with PCNT (gray), TACC3 (magenta), Tubulin (green) and DAPI (blue). Scale bars 10 µm. (D, H, L) Quantification of aMTOC parameters in (A), (G), (K), respectively. (E, I, M) Quantification of spindle and LISD parameters in (A), (G), (K), respectively. Black dots: DMSO-treated; gray dots: MLN-treated. See also Figure S2 which contains statistics.

Article Snippet: Primary antibodies and concentrations were used as follows: Pericentrin (Pcnt) (mouse, 1:100; BD Biosciences, #611814); TACC3 (Rabbit, 1:100; Novus Biologicals # NBP2-67671), Alpha Tubulin polyclonal (Sheep, 1:100; Cytoskeleton #ATN02); phosphorylated ABC (rabbit, 1:100; Cell Signaling Technology, #2914), phosphorylated CDC25B (1:100; Signalway Antibodies #11949); phosphorylated INCENP (Gift from Michael Lampson), Human anti-ACA (1:30, Antibodies Incorporated #15-234).

Techniques: Staining

Journal: bioRxiv

Article Title: Spatio-temporal requirements of Aurora kinase A in mouse oocytes meiotic spindle building

doi: 10.1101/2024.04.01.587547

Figure Lengend Snippet: (A) Live cell light-sheet imaging of WT and BC-MLN-treated KO oocytes during Early pro-metaphase I; aMTOC (gray), SiR-tubulin (green), DNA (magenta). Time is indicated in h:min; Scale bar 10 μm (B, D) Quantification of spindle volume over time in WT (B) and BC-KO (D), respectively. (C, E) Quantification of area and aMTOC numbers over time in WT (C) and BC-KO (E), respectively. Yellow shadow: DMSO-treated; Purple shadow: MLN-treated. The red arrows indicate MLN addition time. Number of oocytes: WT DMSO: 11; WT MLN: 16; BC-KO DMSO:6; BC-KO MLN:8.

Article Snippet: Primary antibodies and concentrations were used as follows: Pericentrin (Pcnt) (mouse, 1:100; BD Biosciences, #611814); TACC3 (Rabbit, 1:100; Novus Biologicals # NBP2-67671), Alpha Tubulin polyclonal (Sheep, 1:100; Cytoskeleton #ATN02); phosphorylated ABC (rabbit, 1:100; Cell Signaling Technology, #2914), phosphorylated CDC25B (1:100; Signalway Antibodies #11949); phosphorylated INCENP (Gift from Michael Lampson), Human anti-ACA (1:30, Antibodies Incorporated #15-234).

Techniques: Imaging

Journal: bioRxiv

Article Title: Spatio-temporal requirements of Aurora kinase A in mouse oocytes meiotic spindle building

doi: 10.1101/2024.04.01.587547

Figure Lengend Snippet: (A) Schematic of the experimental design. ABC-KO oocytes microinjected with different AURKA fusions. Localization of AURKA is green. (B) Representative confocal images of Metaphase I ABC-KO oocytes expressing the indicated fusions. Non-injected ABC-KO oocytes were control. Oocytes were immuno-stained with TACC3 (magenta), tubulin (green), DAPI (blue). Localization of the targeted AURKA fusion is gray. (C) Quantification of spindle and LISD parameters in (B). Gray dots: Non-injected oocytes; pink dots: WT-AURKA; blue dots: MTOC-AURKA + chromatin-AURKA. (D, E) Representative confocal images of Metaphase I ABC-KO oocytes expressing the indicated fusions immuno-stained with tubulin (green), DAPI (DNA) in (D) and PCNT (green), TACC3 (magenta), DAPI (DNA) in (E). Localization of the targeted AURKA fusion is gray. Yellow arrow indicates MTOC-AURKA at kinetochores. Scale bar: 5µm. (F) Quantification of aMTOC parameters. (G) Quantification of spindle and LISD parameters. Gray dots: Non-injected oocytes; pink dots: WT-AURKA; green dots: MTOC-AURKA, and purple dots: chromatin-AURKA. (H) Live cell light-sheet imaging of WT and ABC-KO oocytes. ABC-KO oocytes expresses the indicated fusion, Sir-tubulin (green), DNA (magenta). (G) Time of MT nucleation and (H) time of spindle bipolarization (One-way ANOVA, **** p<0.0001). Data are represented as mean ± SEM. (K) Spindle volume during meiotic maturation. Time stamp (h:min) is relative to GVBD. In brackets are the number of oocytes analyzed in at least 3 independent experiments. See also Figure S3-S6.

Article Snippet: Primary antibodies and concentrations were used as follows: Pericentrin (Pcnt) (mouse, 1:100; BD Biosciences, #611814); TACC3 (Rabbit, 1:100; Novus Biologicals # NBP2-67671), Alpha Tubulin polyclonal (Sheep, 1:100; Cytoskeleton #ATN02); phosphorylated ABC (rabbit, 1:100; Cell Signaling Technology, #2914), phosphorylated CDC25B (1:100; Signalway Antibodies #11949); phosphorylated INCENP (Gift from Michael Lampson), Human anti-ACA (1:30, Antibodies Incorporated #15-234).

Techniques: Expressing, Injection, Control, Staining, Imaging

Journal: Scientific Reports

Article Title: Changes in EGFR activity following CRISPR/Cas9-editing of the EGF binding domain

doi: 10.1038/s41598-026-37579-8

Figure Lengend Snippet: Distribution of EGF and EGFR and phosphorylation status of EGFR in EGF treated cells. ( A ) ICC for EGF and EGFR in untreated cells and cells treated with EGF for 30 min Confocal imaging was done on laser scan confocal Nikon Ti microscope at 100x magnification; staining was done for nuclei (Hoechst 33342), EGF (ab9595 Abcam, Cambridge, United Kingdom) and EGFR (antibody recognizing extracellular domain of human EGFR: epitope 6-273 aa, antibody clone sc-101; Santa Cruz Biotechnology, TX, United States). The scale bars are 50 μm. ( B ) Phosphorylation of EGFR upon treatment with EGF was determined by Western blot. Whole cell lysates were obtained from cells without treatment or 30 min after treatment with recombinant EGF. Three independent experiments were done and imaged using Li-Cor system; representative Western blots are shown. Normalization was done using α-Tubulin as a loading control (Catalog number 926-42213 Li-Cor, NE, United States). Antibodies for phosphorylated EGFR included Y1068 (Catalog number #3777, Cell Signaling, MA, United States), Y1086 (Catalog number #2220S, Cell Signaling, MA, United States) and Y1173 (Catalog number ab32578 Abcam, Cambridge, United Kingdom). ( C ) Western blot quantification was done using software provided by Li-Cor and plotted using Graph Pad Prizm. This bar graph represents relative quantification of phosphorylation of EGFR (for tyrosine positions Y1068, Y1086 and Y1173) for wild type ME180 cells and mutant clone VII11 at 30 min after EGF treatment. To show relative phosphorylation values, the highest phosphorylation signal (that for Tyrosine 1068 in wild type cells) was set as 1. Results are presented as the means ± SD of at least three independent experiments.

Article Snippet: Normalization was done using anti-Beta-Actin monoclonal primary antibody (Catalog number 926-42210 Li-Cor, NE, United States) or anti-Alpha-Tubulin Mouse monoclonal antibody (Catalog number 926-42213 Li-Cor, NE, United States).

Techniques: Phospho-proteomics, Imaging, Microscopy, Staining, Western Blot, Recombinant, Control, Software, Quantitative Proteomics, Mutagenesis

Journal: Cellular and Molecular Life Sciences

Article Title: Cdk5 regulates IP3R1-mediated Ca 2+ dynamics and Ca 2+ -mediated cell proliferation

doi: 10.1007/s00018-022-04515-8

Figure Lengend Snippet: Cdk5 associates with and phosphorylates IP3R1 at Ser 421 . A Cdk5 associates with IP3R1. Lysates of wt and Cdk5 −/− MEFs were subjected to immunoprecipitation (IP) using IP3R1 antibody. The IPs were resolved by 4–20% gradient SDS-PAGE and then immunoblotted for IP3R1 and Cdk5 (left panel). To assess the specificity of the IP3R1 antibody, lysates of wt MEFs were blotted (right panel) with antibody blocked (lane 2) or not blocked (lane 1) with the peptide antigen that was used to raise the antibody. Lanes 3 and 4 represent IP control using normal IgG. B Cdk5 specifically phosphorylates IP3R1 at Ser 421 . Lysates of wt and Cdk5 −/− MEFs were subjected to SDS-PAGE and then immunoblotted for IP3R1 phosphoSer 421 and phosphoThr 799 , IP3R1, Cdk5 and tubulin. Tubulin blot was used as loading control. Representative blots are from one of four independent experiments ( n = 4) showing similar results. Ratios of levels of IP3R1 phosphoSer 421 (middle panel) and phosphoThr 799 (right panel) vs total IP3R were calculated following densitometric analysis of blots using NIH Image J 1.61. Standard deviations were calculated based on the ratios obtained from the four independent sets of experiments. Values from wt MEFs were normalized to 1.0. * p < 0.05. ns: not significant. C Shows the specificity of the IP3R1 phosphoSer 421 antibody. Lysates of wt MEFs depleted of endogenous IP3R1, but expressing exogenous IP3R1 S 421 A (res) were subjected to immunoblotting for IP3R1 phosphoSer 421 , IP3R1, and Cdk5. GAPDH blot was used as loading control

Article Snippet: Mito-tempo and antibodies against Cdk5 (C-8), tubulin (D-10), IP3R1 (E-8), p21 CIP1 (L-17), Prx1 (N-19) and actin (I-19) were from Santa Cruz Biotech (Manassas, VA, USA).

Techniques: Immunoprecipitation, SDS Page, Control, Expressing, Western Blot

Journal: Cellular and Molecular Life Sciences

Article Title: Cdk5 regulates IP3R1-mediated Ca 2+ dynamics and Ca 2+ -mediated cell proliferation

doi: 10.1007/s00018-022-04515-8

Figure Lengend Snippet: IP3R1 loss inhibits the ATP-induced increase in [Ca 2+ ] cyt in Cdk5 −/− MEFs. A Lysates of cells transfected with IP3R1 siRNA #1 or #2 for 48 h were resolved by SDS-PAGE and immunoblotting for IP3R1 and Cdk5. Tubulin blot was used to assess protein loading. Representative blots are from one of three independent experiments showing similar result are shown. B wt and Cdk5 −/− MEFs transfected with IP3R1 siRNA #1 or #2, loaded with Fluo-4 AM, and treated with 1 µM ATP were analyzed for [Ca 2+ ] cyt transients by single-cell Ca 2+ imaging analyses in Ca 2+ -free buffer. Data are means of Ca 2+ signal traces from 20 cells and are from one of three independent experiments showing similar results. [Ca 2+ ] cyt transients were further analyzed by measuring their peak amplitudes ( C ) and calculating the areas under the curve which begin immediately after addition of 1 mM ATP and ends when the Ca 2+ trace goes back to the baseline level. D Values are means ± SEM from three independent experiments ( n = 3). * and **Denote p < 0.05 and p < 0.01, respectively. E Wt and Cdk5 −/− MEFs were co-transfected with the indicated vector and IP3R1 siRNA #2. Cell lysates (40 µg) were resolved by SDS-PAGE and immunoblotted for IP3R1 and Cdk5. GAPDH blot was used as loading control. F Wt and Cdk5 −/− MEFs co-transfected with the indicated vector and siRNA #2 were loaded with Mag-Fluo-4 AM. ER Ca 2+ release following IP3 treatment was measured by spectrofluorometry. Values, which represent the fold change in peak amplitudes, are means ± SEM from three independent experiments ( n = 3). * p < 0.05

Article Snippet: Mito-tempo and antibodies against Cdk5 (C-8), tubulin (D-10), IP3R1 (E-8), p21 CIP1 (L-17), Prx1 (N-19) and actin (I-19) were from Santa Cruz Biotech (Manassas, VA, USA).

Techniques: Transfection, SDS Page, Western Blot, Imaging, Plasmid Preparation, Control

Journal: Frontiers in molecular biosciences

Article Title: Ciliary phenotyping in renal epithelial cells in a cranioectodermal dysplasia patient with WDR35 variants.

doi: 10.3389/fmolb.2023.1285790

Figure Lengend Snippet: FIGURE 2 Renal epithelial cells (URECs) derived from control 1 and CED patient with WDR35 variants presenting a typical cobblestone epithelial cell layer when grown in 2D culture (A). Human URECs following IF imaging using anti-ARL13B (green) for the ciliary membrane, anti-acetylated-tubulin (red) for the ciliary axoneme, PCNT (pink) to mark the base of the cilium, and DAPI (blue) for the nuclei are shown. P, CED patient; C1, control 1; C2, control 2; and C3, control 3 (B). The IF experiments revealed no significant differences in ciliogenesis between the CED patient and the combined controls (C). There was, however, a statistical difference (p < 0.01) between the CED patient and control 1, a sex- and age-matched control (D).

Article Snippet: After fixation with 2% PFA (Sigma), permeabilization with 1% Triton, and blocking with 2% BSA (Sigma), the cells were incubated with primary antibodies targeting acetylated-alpha-tubulin (Acet-Tub) at 1:1000 (mouse monoclonal T6793, Sigma-Aldrich), ADP-ribosylation factor-like protein 13B (ARL13B) at 1:500 (rabbit polyclonal, Proteintech Group), and pericentrin (PCNT) at 1:1000 (mouse monoclonal, Abcam Cambridge), followed by incubation with fluor-labeled secondary antibodies: anti-mouse Alexa Fluor 568, anti-rabbit Alexa Fluor 488, and anti-mouse Alexa Fluor 647 from Thermo Fisher Scientific.

Techniques: Derivative Assay, Control, Imaging, Membrane

Journal: Frontiers in molecular biosciences

Article Title: Ciliary phenotyping in renal epithelial cells in a cranioectodermal dysplasia patient with WDR35 variants.

doi: 10.3389/fmolb.2023.1285790

Figure Lengend Snippet: FIGURE 3 Cilium length, height, and width were analyzed using two antibodies specific for Acet-Tub and ARL13B. Examination of the cilia width (A axis) revealed highly significant differences between the CED patient and controls for both markers (Acet-Tub and ARL13B: p < 0.0001) (A, B). Evaluation of the ciliary height showed no differences between the CED patient and controls (C, D). Analysis of the ciliary length revealed longer cilia in the CED patient as compared to controls for both proteins (Acet-Tub: p < 0.01 and ARL13B: p < 0.05) (E, F).

Article Snippet: After fixation with 2% PFA (Sigma), permeabilization with 1% Triton, and blocking with 2% BSA (Sigma), the cells were incubated with primary antibodies targeting acetylated-alpha-tubulin (Acet-Tub) at 1:1000 (mouse monoclonal T6793, Sigma-Aldrich), ADP-ribosylation factor-like protein 13B (ARL13B) at 1:500 (rabbit polyclonal, Proteintech Group), and pericentrin (PCNT) at 1:1000 (mouse monoclonal, Abcam Cambridge), followed by incubation with fluor-labeled secondary antibodies: anti-mouse Alexa Fluor 568, anti-rabbit Alexa Fluor 488, and anti-mouse Alexa Fluor 647 from Thermo Fisher Scientific.

Techniques:

Journal: Frontiers in molecular biosciences

Article Title: Ciliary phenotyping in renal epithelial cells in a cranioectodermal dysplasia patient with WDR35 variants.

doi: 10.3389/fmolb.2023.1285790

Figure Lengend Snippet: FIGURE 4 Volume measurements of ARL13B, Acet-Tub, and PCNT. The amount of all markers was calculated and revealed a highly significant increase in volume for all three proteins (Acet-Tub, ARL13B, and PCNT: p < 0.0001) (A–C).

Article Snippet: After fixation with 2% PFA (Sigma), permeabilization with 1% Triton, and blocking with 2% BSA (Sigma), the cells were incubated with primary antibodies targeting acetylated-alpha-tubulin (Acet-Tub) at 1:1000 (mouse monoclonal T6793, Sigma-Aldrich), ADP-ribosylation factor-like protein 13B (ARL13B) at 1:500 (rabbit polyclonal, Proteintech Group), and pericentrin (PCNT) at 1:1000 (mouse monoclonal, Abcam Cambridge), followed by incubation with fluor-labeled secondary antibodies: anti-mouse Alexa Fluor 568, anti-rabbit Alexa Fluor 488, and anti-mouse Alexa Fluor 647 from Thermo Fisher Scientific.

Techniques:

Journal: Molecular cell

Article Title: Differentiation drives widespread rewiring of the neural stem cell chaperone network

doi: 10.1016/j.molcel.2020.03.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-Tcp1δ (H-1) , Santa Cruz Biotech. , sc-137092: RRID: AB_2073768.

Techniques: Affinity Purification, Virus, Recombinant, Transfection, Cell Culture, Staining, Flow Cytometry, Fluorescence, Microscopy, Luminescence Assay, Imaging, Plasmid Preparation, Software

Journal: Applied Nanoscience

Article Title: Detection of heavy metals (Cu+2, Hg+2) by biosynthesized silver nanoparticles

doi: 10.1007/s13204-015-0452-4

Figure Lengend Snippet: Fig. 5 TEM micrographs of Cu-complex with AgNP (A, B, C) and SAED image (inset of C) from C

Article Snippet: The absorbance spectra of the AgNP were analyzed using a ‘SHIMADZU’ UV 1800 spectrophotometer and TEM images were taken using JEOL-JEM 2100 high-resolution transmission electron microscope (HRTEM).

Techniques:

Journal: Applied Nanoscience

Article Title: Detection of heavy metals (Cu+2, Hg+2) by biosynthesized silver nanoparticles

doi: 10.1007/s13204-015-0452-4

Figure Lengend Snippet: Fig. 4 TEM micrographs of AgNP (A, B) and SAED (C) image from (B)

Article Snippet: The absorbance spectra of the AgNP were analyzed using a ‘SHIMADZU’ UV 1800 spectrophotometer and TEM images were taken using JEOL-JEM 2100 high-resolution transmission electron microscope (HRTEM).

Techniques:

Journal:

Article Title: In Vivo Imaging of Bioluminescent Escherichia coli in a Cutaneous Wound Infection Model for Evaluation of an Antibiotic Therapy

doi: 10.1128/AAC.48.9.3436-3441.2004

Figure Lengend Snippet: Bacterial strain, plasmids, and primers used in the study

Article Snippet: Reactions were carried out in 50-μl volumes containing 5 μl of 10× PCR buffer (supplied with Taq DNA polymerase), 50 pmol of each oligonucleotide primer, each deoxynucleotide triphosphate (dATP, dCTP, dGTP, and dTTP) at a concentration of 0.2 mM, 1 U of Taq DNA polymerase, and 10 ng of plasmid DNA containing firefly luciferase (pSP luc ). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain, plasmid, or primer Genotype or sequence Source or reference E. coli TOP10F′ Δ( ara - leu ) 7697 galU galK rpsL endA1 nupG Invitrogen (NO-C615-00) Plasmids pSP-luc Amp r Ori colE1 Promega (E1781) pRB474 Amp r Cm r Ori − Ori + 6 Primers LucF (5′-3′) AGG AA G GAT CC A GAG CAG ATT GTA CTG AGA G a This study LucR (5′-3′) AAT GCA GGT TAA CCT GGC TTA TCG AAA TT Open in a separate window a Underlined nucleotides show the position of the BamHI restriction site.

Techniques: Plasmid Preparation, Sequencing, Acetylene Reduction Assay

Journal:

Article Title: In Vivo Imaging of Bioluminescent Escherichia coli in a Cutaneous Wound Infection Model for Evaluation of an Antibiotic Therapy

doi: 10.1128/AAC.48.9.3436-3441.2004

Figure Lengend Snippet: Emission of light by E. coli cells as a function of storage time in Luria broth at two different temperatures (20 and 30°C). RLU, relative light units.

Article Snippet: Reactions were carried out in 50-μl volumes containing 5 μl of 10× PCR buffer (supplied with Taq DNA polymerase), 50 pmol of each oligonucleotide primer, each deoxynucleotide triphosphate (dATP, dCTP, dGTP, and dTTP) at a concentration of 0.2 mM, 1 U of Taq DNA polymerase, and 10 ng of plasmid DNA containing firefly luciferase (pSP luc ). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain, plasmid, or primer Genotype or sequence Source or reference E. coli TOP10F′ Δ( ara - leu ) 7697 galU galK rpsL endA1 nupG Invitrogen (NO-C615-00) Plasmids pSP-luc Amp r Ori colE1 Promega (E1781) pRB474 Amp r Cm r Ori − Ori + 6 Primers LucF (5′-3′) AGG AA G GAT CC A GAG CAG ATT GTA CTG AGA G a This study LucR (5′-3′) AAT GCA GGT TAA CCT GGC TTA TCG AAA TT Open in a separate window a Underlined nucleotides show the position of the BamHI restriction site.

Techniques:

Journal:

Article Title: In Vivo Imaging of Bioluminescent Escherichia coli in a Cutaneous Wound Infection Model for Evaluation of an Antibiotic Therapy

doi: 10.1128/AAC.48.9.3436-3441.2004

Figure Lengend Snippet: Effects of increasing concentrations of SMX-TMP on the light emission (relative light units) of E. coli detected with the bioluminescence imaging system. Each concentration of SMX-TMP (0.3, 0.6, 0.9, and 1.2 mg/liter) was incubated with the bioluminescent E. coli for 30 min (▪) and 4 h (□) at 35°C in Luria broth. The sample labeled “Control” is the untreated strain.

Article Snippet: Reactions were carried out in 50-μl volumes containing 5 μl of 10× PCR buffer (supplied with Taq DNA polymerase), 50 pmol of each oligonucleotide primer, each deoxynucleotide triphosphate (dATP, dCTP, dGTP, and dTTP) at a concentration of 0.2 mM, 1 U of Taq DNA polymerase, and 10 ng of plasmid DNA containing firefly luciferase (pSP luc ). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain, plasmid, or primer Genotype or sequence Source or reference E. coli TOP10F′ Δ( ara - leu ) 7697 galU galK rpsL endA1 nupG Invitrogen (NO-C615-00) Plasmids pSP-luc Amp r Ori colE1 Promega (E1781) pRB474 Amp r Cm r Ori − Ori + 6 Primers LucF (5′-3′) AGG AA G GAT CC A GAG CAG ATT GTA CTG AGA G a This study LucR (5′-3′) AAT GCA GGT TAA CCT GGC TTA TCG AAA TT Open in a separate window a Underlined nucleotides show the position of the BamHI restriction site.

Techniques: Imaging, Concentration Assay, Incubation, Labeling, Control

Journal:

Article Title: In Vivo Imaging of Bioluminescent Escherichia coli in a Cutaneous Wound Infection Model for Evaluation of an Antibiotic Therapy

doi: 10.1128/AAC.48.9.3436-3441.2004

Figure Lengend Snippet: In vivo monitoring of E. coli(pRB474) bioluminescence in the cutaneous wound by use of the bioluminescence imaging system. Each set of data represents the mean (±standard deviation |) number of relative light units (RLU) for three hairless rats either untreated (♦) or treated with SMX-TMP (▪; 30 mg/kg) and imaged for 5 min at 0, 4, 8, 24, 36, and 48 h postinfection. *, statistically different by the Student t test.

Article Snippet: Reactions were carried out in 50-μl volumes containing 5 μl of 10× PCR buffer (supplied with Taq DNA polymerase), 50 pmol of each oligonucleotide primer, each deoxynucleotide triphosphate (dATP, dCTP, dGTP, and dTTP) at a concentration of 0.2 mM, 1 U of Taq DNA polymerase, and 10 ng of plasmid DNA containing firefly luciferase (pSP luc ). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain, plasmid, or primer Genotype or sequence Source or reference E. coli TOP10F′ Δ( ara - leu ) 7697 galU galK rpsL endA1 nupG Invitrogen (NO-C615-00) Plasmids pSP-luc Amp r Ori colE1 Promega (E1781) pRB474 Amp r Cm r Ori − Ori + 6 Primers LucF (5′-3′) AGG AA G GAT CC A GAG CAG ATT GTA CTG AGA G a This study LucR (5′-3′) AAT GCA GGT TAA CCT GGC TTA TCG AAA TT Open in a separate window a Underlined nucleotides show the position of the BamHI restriction site.

Techniques: In Vivo, Imaging, Standard Deviation

Journal:

Article Title: In Vivo Imaging of Bioluminescent Escherichia coli in a Cutaneous Wound Infection Model for Evaluation of an Antibiotic Therapy

doi: 10.1128/AAC.48.9.3436-3441.2004

Figure Lengend Snippet: Monitoring of the effects of SMX-TMP on bioluminescent E. coli(pRB474) in a hairless rat. Bioluminescent bacteria were injected into the wound (5 × 107 CFU). Shown is one treated (SMX-TMP) E. coli(pRB474)-infected rat (right set of panels) and one untreated E. coli(pRB474)-infected rat (left set of panels) imaged dorsally for 5 min at 4, 24, and 48 h postinfection.

Article Snippet: Reactions were carried out in 50-μl volumes containing 5 μl of 10× PCR buffer (supplied with Taq DNA polymerase), 50 pmol of each oligonucleotide primer, each deoxynucleotide triphosphate (dATP, dCTP, dGTP, and dTTP) at a concentration of 0.2 mM, 1 U of Taq DNA polymerase, and 10 ng of plasmid DNA containing firefly luciferase (pSP luc ). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain, plasmid, or primer Genotype or sequence Source or reference E. coli TOP10F′ Δ( ara - leu ) 7697 galU galK rpsL endA1 nupG Invitrogen (NO-C615-00) Plasmids pSP-luc Amp r Ori colE1 Promega (E1781) pRB474 Amp r Cm r Ori − Ori + 6 Primers LucF (5′-3′) AGG AA G GAT CC A GAG CAG ATT GTA CTG AGA G a This study LucR (5′-3′) AAT GCA GGT TAA CCT GGC TTA TCG AAA TT Open in a separate window a Underlined nucleotides show the position of the BamHI restriction site.

Techniques: Bacteria, Injection, Infection