e coli dh5a competent cells  (Millipore)


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    Escherichia coli
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    Structured Review

    Millipore e coli dh5a competent cells

    https://www.bioz.com/result/e coli dh5a competent cells/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Clone Assay:

    Article Title: Transgenic Hybrid Poplar for Sustainable and Scalable Production of the Commodity/Specialty Chemical, 2-Phenylethanol
    Article Snippet: .. Heterologous Expression in E. coli RhPAAS and PAR1 genes were cloned in tandem into a pETDuet-1 expression vector (Novagen) as follows. .. PAR1 cDNA was first PCR amplified using primers PAR1-Nde I-DUET-FOR and PAR1-Xho I-DUET-REV ( ) to include Nde I and Xho I restriction enzyme sites at the 5′- and 3′-ends, respectively, then cloned into a pCR4-TOPO vector and excised and then cloned into the MCS2 region of the pETDuet-1 vector.

    Positive Control:

    Article Title: The antimicrobial molecule trappin-2/elafin has anti-parasitic properties and is protective in vivo in a murine model of cerebral malaria
    Article Snippet: .. As a positive control for the ELISA, the supernatant from MNs stimulated with 0.2 μg/ml of LPS (Escherichia coli 0127:B8, from Sigma) was used for T-2 assessment. .. Parasite and T-2 Immunolocalisation assays After preparation of PVM-enclosed merozoite structures (merozoites “bags”) from which the egress of malaria parasites was checked every 1–2 hrs, they were incubated for 2 h with 50 μg/ml of recombinant human T-2 (Sigma).

    Cell Culture:

    Article Title: Control of siRNA expression using the Cre-loxP recombination system
    Article Snippet: .. For residual E.coli lysate, pTriEx-3 Hygro empty vector was introduced into E.coli strain RosettaBlue(DE3)pLacI (Novagen) and cultured in LB medium that contained 1% glucose, 34 µg/ml chloramphenicol, 12.5 µg/ml tetracycline and 100 µg/ml ampicillin at 37°C until absorbance at 600 nm reached 0.7. .. After addition of IPTG to a final concentration of 1 mM, E.coli were harvested and treated in the same way as that of pTriEx-3 Hygro-TAT-NLS-Cre or pTriEx-3 Hygro-TAT-NLS-EGFP.

    Purification:

    Article Title: MYCN acts as a direct co-regulator of p53 in MYCN amplified neuroblastoma
    Article Snippet: .. BL-21 E.Coli cells expressing MYCN-6×His were lysed in His lysis buffer (50 mM NaH2PO4, 1% Triton, 1 μg/μl lysozyme, 1 mM PMSF, 300 mM NaCl, and 20 mM imidazole), purified using HIS-Select Nickel Affinity Gel (Sigma Aldrich) and eluted with 350 mM imidazole in His lysis buffer without lysozyme. .. Purified MYCN-6×His protein was incubated with glutathione beads coated with equimolar amounts of either purified GST or GST-p53 overnight at 4°C.

    Enzyme-linked Immunosorbent Assay:

    Article Title: The antimicrobial molecule trappin-2/elafin has anti-parasitic properties and is protective in vivo in a murine model of cerebral malaria
    Article Snippet: .. As a positive control for the ELISA, the supernatant from MNs stimulated with 0.2 μg/ml of LPS (Escherichia coli 0127:B8, from Sigma) was used for T-2 assessment. .. Parasite and T-2 Immunolocalisation assays After preparation of PVM-enclosed merozoite structures (merozoites “bags”) from which the egress of malaria parasites was checked every 1–2 hrs, they were incubated for 2 h with 50 μg/ml of recombinant human T-2 (Sigma).

    Incubation:

    Article Title: U-Bang-Haequi Tang: A Herbal Prescription that Prevents Acute Inflammation through Inhibition of NF- κB-Mediated Inducible Nitric Oxide Synthase
    Article Snippet: .. Raw264.7 cells were incubated with 1 μ g/mL LPS (Escherichia coli 026:B6; Sigma, St. Louis, MO, USA). .. The cells were incubated without 10% fetal bovine serum for 24 h and then stimulated to LPS or LPS + UBT for the indicated time periods.

    Expressing:

    Article Title: Transgenic Hybrid Poplar for Sustainable and Scalable Production of the Commodity/Specialty Chemical, 2-Phenylethanol
    Article Snippet: .. Heterologous Expression in E. coli RhPAAS and PAR1 genes were cloned in tandem into a pETDuet-1 expression vector (Novagen) as follows. .. PAR1 cDNA was first PCR amplified using primers PAR1-Nde I-DUET-FOR and PAR1-Xho I-DUET-REV ( ) to include Nde I and Xho I restriction enzyme sites at the 5′- and 3′-ends, respectively, then cloned into a pCR4-TOPO vector and excised and then cloned into the MCS2 region of the pETDuet-1 vector.

    Article Title: Comparison of the functional properties of trimeric and monomeric CaiT of Escherichia coli
    Article Snippet: .. E. coli JW0039 (F− ΔcaiT753::kan Δ (araD-araB )567 ΔlacZ4787 (::rrnB-3) λ − rph-1 Δ (rhaD-rhaB )568 hsdR514 ) or E. coli BL21(DE3) pLysS (F− ompT hasdSB (rB− mB− ) gal dcm (DE3) pLysS (camR )) (Novagen) harboring given plasmids was used for expression of the caiT gene of E. coli and biochemical assays. .. For the pulse-chase experiments, E. coli WG170 (F− trp lacZ rpsL thi Δ (putPA )101 proP219 ) was used.

    Article Title: MYCN acts as a direct co-regulator of p53 in MYCN amplified neuroblastoma
    Article Snippet: .. BL-21 E.Coli cells expressing MYCN-6×His were lysed in His lysis buffer (50 mM NaH2PO4, 1% Triton, 1 μg/μl lysozyme, 1 mM PMSF, 300 mM NaCl, and 20 mM imidazole), purified using HIS-Select Nickel Affinity Gel (Sigma Aldrich) and eluted with 350 mM imidazole in His lysis buffer without lysozyme. .. Purified MYCN-6×His protein was incubated with glutathione beads coated with equimolar amounts of either purified GST or GST-p53 overnight at 4°C.

    Article Title: Human RNA lariat debranching enzyme cDNA complements the phenotypes of Saccharomyces cerevisiae dbr1 and Schizosaccharomyces pombe dbr1 mutants
    Article Snippet: .. For construction of E.coli expression plasmid pKN203 a Nde I– Bam HI full-length hDBR1 cDNA fragment from pKN201 was subcloned into Nde I and Bam HI digested pET28a (Novagen). .. The yeast expression plasmids pKN205 and pKN206 were constructed by inserting an Eco RI– Bam HI full-length hDBR1 cDNA fragment from pKN201 into S.cerevisiae expression vectors pRS314GU ( ) and pRS316GU ( ) digested with Eco RI and Bam HI, respectively.

    Lysis:

    Article Title: MYCN acts as a direct co-regulator of p53 in MYCN amplified neuroblastoma
    Article Snippet: .. BL-21 E.Coli cells expressing MYCN-6×His were lysed in His lysis buffer (50 mM NaH2PO4, 1% Triton, 1 μg/μl lysozyme, 1 mM PMSF, 300 mM NaCl, and 20 mM imidazole), purified using HIS-Select Nickel Affinity Gel (Sigma Aldrich) and eluted with 350 mM imidazole in His lysis buffer without lysozyme. .. Purified MYCN-6×His protein was incubated with glutathione beads coated with equimolar amounts of either purified GST or GST-p53 overnight at 4°C.

    Plasmid Preparation:

    Article Title: Transgenic Hybrid Poplar for Sustainable and Scalable Production of the Commodity/Specialty Chemical, 2-Phenylethanol
    Article Snippet: .. Heterologous Expression in E. coli RhPAAS and PAR1 genes were cloned in tandem into a pETDuet-1 expression vector (Novagen) as follows. .. PAR1 cDNA was first PCR amplified using primers PAR1-Nde I-DUET-FOR and PAR1-Xho I-DUET-REV ( ) to include Nde I and Xho I restriction enzyme sites at the 5′- and 3′-ends, respectively, then cloned into a pCR4-TOPO vector and excised and then cloned into the MCS2 region of the pETDuet-1 vector.

    Article Title: Human RNA lariat debranching enzyme cDNA complements the phenotypes of Saccharomyces cerevisiae dbr1 and Schizosaccharomyces pombe dbr1 mutants
    Article Snippet: .. For construction of E.coli expression plasmid pKN203 a Nde I– Bam HI full-length hDBR1 cDNA fragment from pKN201 was subcloned into Nde I and Bam HI digested pET28a (Novagen). .. The yeast expression plasmids pKN205 and pKN206 were constructed by inserting an Eco RI– Bam HI full-length hDBR1 cDNA fragment from pKN201 into S.cerevisiae expression vectors pRS314GU ( ) and pRS316GU ( ) digested with Eco RI and Bam HI, respectively.

    Article Title: Control of siRNA expression using the Cre-loxP recombination system
    Article Snippet: .. For residual E.coli lysate, pTriEx-3 Hygro empty vector was introduced into E.coli strain RosettaBlue(DE3)pLacI (Novagen) and cultured in LB medium that contained 1% glucose, 34 µg/ml chloramphenicol, 12.5 µg/ml tetracycline and 100 µg/ml ampicillin at 37°C until absorbance at 600 nm reached 0.7. .. After addition of IPTG to a final concentration of 1 mM, E.coli were harvested and treated in the same way as that of pTriEx-3 Hygro-TAT-NLS-Cre or pTriEx-3 Hygro-TAT-NLS-EGFP.

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  • 99
    Millipore hygromycin b
    Hygromycin B, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 574 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hygromycin b/product/Millipore
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    92
    Millipore escherichia coli cells
    Overmethylation alleviates the highly restrictive phenotype and suppresses the autorestriction defects. <t>Escherichia</t> coli ER1992 (A) or MP060 cells (B, C) carrying pACYCeco or pIM-RM plasmids were co-transformed with pBADecoM carrying the arabinose inducible ecoRIM gene. MTase expression was induced with 0.04% for 1 h at 37 °C (A) or overnight (B, C). Then, a quantitative assay for restriction λ vir phage DNA was performed (A), and SOS-inducing YFP fluorescence was measured (B) or microscopy of cells was conducted (C).
    Escherichia Coli Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/escherichia coli cells/product/Millipore
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    90
    Millipore e chaffeensis infected thp 1 cells
    Growth of E. <t>chaffeensis</t> is reversibly inhibited by the class III PtdIns3K inhibitor 3-MA. (A) 3-MA does not inhibit internalization of E. chaffeensis . The percentage of intracellular bacteria vs. total cell-associated bacteria was determined in <t>THP-1</t> cells incubated with 3-MA or RPMI 1640 medium control (RPMI) at 2 h p.i. by scoring 100 E. chaffeensis bacteria in each group after 2 rounds of immunofluorescence labeling. Data are presented as the mean ± standard deviation of triplicate samples (not significantly different by the Student t test, P > 0.05). (B and C) 3-MA added at 1 h or 1 d p.i. inhibits E. chaffeensis infection. 3-MA was added to THP-1 cells at a final concentration of 2 mM, and infection was assessed at 3 d p.i. by Diff-Quik staining to determine the percentage of infected cells (B) and the number of bacteria per cell (C). Data are presented as the mean ± standard deviation of triplicate assays. *, Significantly different by the Tukey HSD test ( P
    E Chaffeensis Infected Thp 1 Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e chaffeensis infected thp 1 cells/product/Millipore
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    Overmethylation alleviates the highly restrictive phenotype and suppresses the autorestriction defects. Escherichia coli ER1992 (A) or MP060 cells (B, C) carrying pACYCeco or pIM-RM plasmids were co-transformed with pBADecoM carrying the arabinose inducible ecoRIM gene. MTase expression was induced with 0.04% for 1 h at 37 °C (A) or overnight (B, C). Then, a quantitative assay for restriction λ vir phage DNA was performed (A), and SOS-inducing YFP fluorescence was measured (B) or microscopy of cells was conducted (C).

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    Article Title: Low-level expression of the Type II restriction–modification system confers potent bacteriophage resistance in Escherichia coli

    doi: 10.1093/dnares/dsaa003

    Figure Lengend Snippet: Overmethylation alleviates the highly restrictive phenotype and suppresses the autorestriction defects. Escherichia coli ER1992 (A) or MP060 cells (B, C) carrying pACYCeco or pIM-RM plasmids were co-transformed with pBADecoM carrying the arabinose inducible ecoRIM gene. MTase expression was induced with 0.04% for 1 h at 37 °C (A) or overnight (B, C). Then, a quantitative assay for restriction λ vir phage DNA was performed (A), and SOS-inducing YFP fluorescence was measured (B) or microscopy of cells was conducted (C).

    Article Snippet: Escherichia coli cells were grown in LB medium with antibiotics at the following concentrations when necessary: ampicillin (Ap) at 100 μg/ml, chloramphenicol (Cm) at 30 μg/ml, and tetracycline (Tc) 15 μg/ml.

    Techniques: Transformation Assay, Expressing, Fluorescence, Microscopy

    Phage restriction variation in Escherichia coli cells carrying the EcoRI R–M system measured as phage λ vir infection efficiency. (A) The qualitative assay involved 5 μl of serial dilutions of λb2 vir phage, which were spotted onto LB agar plates with an E. coli ER1992 lawn carrying the indicated plasmids or no plasmids [ER1992E (EcoRI R–M) + strain] and incubated overnight at 37 °C. Quantitative results of the restriction activity of E. coli ER1992 strains. EOP, efficiency of plaques, PFUs tested on plasmid/strains divided by PFUs on pACYC184; and RR, restriction relative to the pIM-RM bearing strain. The standard deviation from at least three measurements is indicated. (B) Western blots of the lysates of the late exponential phase harvested MG1655 bacteria carrying: pACYCeco, pIM-RM, pBAD-RM, and pACYCΔnutL. Lanes R contain a purified R.EcoRI preparation. Lane ‘sm’ contains molecular size markers. The levels of R.EcoRI and M.EcoRI proteins in bacterial crude extracts were tested by western blotting using rabbit anti-M.EcoRI and anti-R.EcoRI polyclonal antibodies and visualized by BCIP/NBT (nitroblue tetrazolium) as the color development reagent. The star and white circles indicate the location of unknown antigen proteins. Note that expression of the EcoRI R–M system on pBAD-RM was induced using 0.04% l -arabinose for 2 h. (C) Relative strength of the P L1 promoter and nutL /N anti-termination in pACYCeco expressed as reporter LacZ activity. Error bars represent standard deviations from at least three independent experiments. (D) The anti-termination-mediated increase of ecoRIR expression from pACYCeco plasmid, by λ-N protein, 10, 20, 30, 40, and 60 min after infection. Note that the cell lysis was observed after 60 min of post-infection time (Line 8), thus the bacterial lysate might be non-representative. Also shown is the comparison of the expression levels of the ecoRIRM from pIM-RM after 30 min of λ vir phage infection. The star indicates the location of the unknown antigen protein, and the arrow indicates the position of the R.EcoRI enzyme.

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    Article Title: Low-level expression of the Type II restriction–modification system confers potent bacteriophage resistance in Escherichia coli

    doi: 10.1093/dnares/dsaa003

    Figure Lengend Snippet: Phage restriction variation in Escherichia coli cells carrying the EcoRI R–M system measured as phage λ vir infection efficiency. (A) The qualitative assay involved 5 μl of serial dilutions of λb2 vir phage, which were spotted onto LB agar plates with an E. coli ER1992 lawn carrying the indicated plasmids or no plasmids [ER1992E (EcoRI R–M) + strain] and incubated overnight at 37 °C. Quantitative results of the restriction activity of E. coli ER1992 strains. EOP, efficiency of plaques, PFUs tested on plasmid/strains divided by PFUs on pACYC184; and RR, restriction relative to the pIM-RM bearing strain. The standard deviation from at least three measurements is indicated. (B) Western blots of the lysates of the late exponential phase harvested MG1655 bacteria carrying: pACYCeco, pIM-RM, pBAD-RM, and pACYCΔnutL. Lanes R contain a purified R.EcoRI preparation. Lane ‘sm’ contains molecular size markers. The levels of R.EcoRI and M.EcoRI proteins in bacterial crude extracts were tested by western blotting using rabbit anti-M.EcoRI and anti-R.EcoRI polyclonal antibodies and visualized by BCIP/NBT (nitroblue tetrazolium) as the color development reagent. The star and white circles indicate the location of unknown antigen proteins. Note that expression of the EcoRI R–M system on pBAD-RM was induced using 0.04% l -arabinose for 2 h. (C) Relative strength of the P L1 promoter and nutL /N anti-termination in pACYCeco expressed as reporter LacZ activity. Error bars represent standard deviations from at least three independent experiments. (D) The anti-termination-mediated increase of ecoRIR expression from pACYCeco plasmid, by λ-N protein, 10, 20, 30, 40, and 60 min after infection. Note that the cell lysis was observed after 60 min of post-infection time (Line 8), thus the bacterial lysate might be non-representative. Also shown is the comparison of the expression levels of the ecoRIRM from pIM-RM after 30 min of λ vir phage infection. The star indicates the location of the unknown antigen protein, and the arrow indicates the position of the R.EcoRI enzyme.

    Article Snippet: Escherichia coli cells were grown in LB medium with antibiotics at the following concentrations when necessary: ampicillin (Ap) at 100 μg/ml, chloramphenicol (Cm) at 30 μg/ml, and tetracycline (Tc) 15 μg/ml.

    Techniques: Infection, Incubation, Activity Assay, Plasmid Preparation, Standard Deviation, Western Blot, Purification, Expressing, Lysis

    The SOS response induction and R–M system instability in cells carrying the plasmid with the high R–M genes expression. (A) Escherichia coli strain SS996, where the gfp gene is under the control of the P sulA promoter, was transformed with the following plasmids: pIM-RM (high R–M genes expression), pACYCeco (low R–M genes expression) and pBAD-RM, where REase expression was induced by 0.04% of l -arabinose for 2 h. Light and fluorescence microscopy images were captured. (B) The SOS response was also measured quantitatively for the same plasmids in E. coli strains with the YFP reporter gene under the control of the P sulA promoter in the background of recA + (MP060) and recA − (MP064). As a control, the plasmid, pIM-RM, with a kanamycin resistance cassette inserted in cat gene upstream of the P R promoter for REase gene (pIM-RMkan) was used, as well as strains without plasmids. (C) Plasmids pIM-RM and pACYCeco (both restriction proficient R + M + ; high and low REase producers) were challenged using a 3-day growth competition assay to generate the co-cultures with the MG1655 strain without a plasmid (no RM system). As a control, plasmid with a restriction-negative variant was also used (pIM27: R − M + ). Briefly, strains without plasmid were mixed 1:1 with cells carrying plasmid with the R–M system variant and allowed to grow in the co-cultures for 60 generations. Chloramphenicol resistance was used as a selection marker for CFUs of cells carrying the R–M system variants. The cell viability was measured at Generations 1 and 60, and calculated as the ratio of the CFUs on LB agar supplemented with chloramphenicol to the CFUs obtained on LB agar.

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    Article Title: Low-level expression of the Type II restriction–modification system confers potent bacteriophage resistance in Escherichia coli

    doi: 10.1093/dnares/dsaa003

    Figure Lengend Snippet: The SOS response induction and R–M system instability in cells carrying the plasmid with the high R–M genes expression. (A) Escherichia coli strain SS996, where the gfp gene is under the control of the P sulA promoter, was transformed with the following plasmids: pIM-RM (high R–M genes expression), pACYCeco (low R–M genes expression) and pBAD-RM, where REase expression was induced by 0.04% of l -arabinose for 2 h. Light and fluorescence microscopy images were captured. (B) The SOS response was also measured quantitatively for the same plasmids in E. coli strains with the YFP reporter gene under the control of the P sulA promoter in the background of recA + (MP060) and recA − (MP064). As a control, the plasmid, pIM-RM, with a kanamycin resistance cassette inserted in cat gene upstream of the P R promoter for REase gene (pIM-RMkan) was used, as well as strains without plasmids. (C) Plasmids pIM-RM and pACYCeco (both restriction proficient R + M + ; high and low REase producers) were challenged using a 3-day growth competition assay to generate the co-cultures with the MG1655 strain without a plasmid (no RM system). As a control, plasmid with a restriction-negative variant was also used (pIM27: R − M + ). Briefly, strains without plasmid were mixed 1:1 with cells carrying plasmid with the R–M system variant and allowed to grow in the co-cultures for 60 generations. Chloramphenicol resistance was used as a selection marker for CFUs of cells carrying the R–M system variants. The cell viability was measured at Generations 1 and 60, and calculated as the ratio of the CFUs on LB agar supplemented with chloramphenicol to the CFUs obtained on LB agar.

    Article Snippet: Escherichia coli cells were grown in LB medium with antibiotics at the following concentrations when necessary: ampicillin (Ap) at 100 μg/ml, chloramphenicol (Cm) at 30 μg/ml, and tetracycline (Tc) 15 μg/ml.

    Techniques: Plasmid Preparation, Expressing, Transformation Assay, Fluorescence, Microscopy, Competitive Binding Assay, Variant Assay, Selection, Marker

    Growth of E. chaffeensis is reversibly inhibited by the class III PtdIns3K inhibitor 3-MA. (A) 3-MA does not inhibit internalization of E. chaffeensis . The percentage of intracellular bacteria vs. total cell-associated bacteria was determined in THP-1 cells incubated with 3-MA or RPMI 1640 medium control (RPMI) at 2 h p.i. by scoring 100 E. chaffeensis bacteria in each group after 2 rounds of immunofluorescence labeling. Data are presented as the mean ± standard deviation of triplicate samples (not significantly different by the Student t test, P > 0.05). (B and C) 3-MA added at 1 h or 1 d p.i. inhibits E. chaffeensis infection. 3-MA was added to THP-1 cells at a final concentration of 2 mM, and infection was assessed at 3 d p.i. by Diff-Quik staining to determine the percentage of infected cells (B) and the number of bacteria per cell (C). Data are presented as the mean ± standard deviation of triplicate assays. *, Significantly different by the Tukey HSD test ( P

    Journal: Autophagy

    Article Title: Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase

    doi: 10.1080/15548627.2016.1217369

    Figure Lengend Snippet: Growth of E. chaffeensis is reversibly inhibited by the class III PtdIns3K inhibitor 3-MA. (A) 3-MA does not inhibit internalization of E. chaffeensis . The percentage of intracellular bacteria vs. total cell-associated bacteria was determined in THP-1 cells incubated with 3-MA or RPMI 1640 medium control (RPMI) at 2 h p.i. by scoring 100 E. chaffeensis bacteria in each group after 2 rounds of immunofluorescence labeling. Data are presented as the mean ± standard deviation of triplicate samples (not significantly different by the Student t test, P > 0.05). (B and C) 3-MA added at 1 h or 1 d p.i. inhibits E. chaffeensis infection. 3-MA was added to THP-1 cells at a final concentration of 2 mM, and infection was assessed at 3 d p.i. by Diff-Quik staining to determine the percentage of infected cells (B) and the number of bacteria per cell (C). Data are presented as the mean ± standard deviation of triplicate assays. *, Significantly different by the Tukey HSD test ( P

    Article Snippet: Co-immunoprecipitation Uninfected or E. chaffeensis -infected THP-1 cells at 2 d p.i. were lysed in modified lysis buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 1% NP40 [USB, 19638], 5% glycerol, and 1% protease inhibitor cocktail III [Calbiochem, 539134]) for 15 min and immunoprecipitated for 2 h with rabbit anti-Etf-1, -BECN1, or -PIK3C3, or mouse anti-RAB5 IgG that was cross-linked to protein A/G-magnetic beads (Pierce, 88805).

    Techniques: Incubation, Immunofluorescence, Labeling, Standard Deviation, Infection, Concentration Assay, Diff-Quik, Staining

    Etf-1 interacts with the RAB5-class III PtdIns3K complex. Co-immunoprecipitation of native Etf-1 with endogenous RAB5, BECN1, and PIK3C3/VPS34. Uninfected (THP) or E. chaffeensis -infected ( Ech ) THP-1 cells at 2 d p.i. were lysed in modified lysis buffer and immunoprecipitated ( IP ) with rabbit anti-Etf-1, BECN1, or PIK3C3, or mouse anti-RAB5 IgG cross-linked to protein A/G-magnetic beads for 2 h. Bound proteins were eluted and subjected to western blotting. Arrows indicate the target proteins. *, IgG heavy or light chains. The number under the figure is the density ratio relative to ACT/actin, with uninfected cells set as 1. The absence of a number indicates an infinite ratio (i.e., the protein was absent in the control). Images were representative of 3 experiments with similar results.

    Journal: Autophagy

    Article Title: Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase

    doi: 10.1080/15548627.2016.1217369

    Figure Lengend Snippet: Etf-1 interacts with the RAB5-class III PtdIns3K complex. Co-immunoprecipitation of native Etf-1 with endogenous RAB5, BECN1, and PIK3C3/VPS34. Uninfected (THP) or E. chaffeensis -infected ( Ech ) THP-1 cells at 2 d p.i. were lysed in modified lysis buffer and immunoprecipitated ( IP ) with rabbit anti-Etf-1, BECN1, or PIK3C3, or mouse anti-RAB5 IgG cross-linked to protein A/G-magnetic beads for 2 h. Bound proteins were eluted and subjected to western blotting. Arrows indicate the target proteins. *, IgG heavy or light chains. The number under the figure is the density ratio relative to ACT/actin, with uninfected cells set as 1. The absence of a number indicates an infinite ratio (i.e., the protein was absent in the control). Images were representative of 3 experiments with similar results.

    Article Snippet: Co-immunoprecipitation Uninfected or E. chaffeensis -infected THP-1 cells at 2 d p.i. were lysed in modified lysis buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 1% NP40 [USB, 19638], 5% glycerol, and 1% protease inhibitor cocktail III [Calbiochem, 539134]) for 15 min and immunoprecipitated for 2 h with rabbit anti-Etf-1, -BECN1, or -PIK3C3, or mouse anti-RAB5 IgG that was cross-linked to protein A/G-magnetic beads (Pierce, 88805).

    Techniques: Immunoprecipitation, Infection, Modification, Lysis, Magnetic Beads, Western Blot, Activated Clotting Time Assay

    E. chaffeensis inclusion membrane is enriched with PtdIns3P and class III PtdIns3K. (A and B) E. chaffeensis ( Ech ) -infected RF/6A cells were transfected with plasmids encoding 2×FYVE-GFP or FLAG-PIK3C3/VPS34. At 15 h p.t. (2 d p.i.), cells were fixed and stained with DAPI to indicate E. chaffeensis (pseudocolored in red). PIK3C3 was labeled with mouse anti-FLAG. Merged/DIC, fluorescence image merged with differential interference contrast (DIC) image. Each boxed area is enlarged 4-fold on the right. N, nucleus; scale bars: 10 μm. (C) The percent colocalization of E. chaffeensis inclusions with PIK3C3 or 2×FYVE was determined by counting 10 to 20 inclusions per cell in 5 to 10 cells per experiment from 3 independent experiments. (D) PtdIns3P levels are increased in E. chaffeensis -infected THP-1 cells. Uninfected or E. chaffeensis -infected THP-1 cells (2 × 10 6 cells) at 1 d p.i. were collected, and PtdIns3P lipids were purified and the amount determined by competitive ELISA. Assays were carried out in triplicate. Data are presented as the mean ± standard deviation. * Significantly different by the Student t test ( P

    Journal: Autophagy

    Article Title: Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase

    doi: 10.1080/15548627.2016.1217369

    Figure Lengend Snippet: E. chaffeensis inclusion membrane is enriched with PtdIns3P and class III PtdIns3K. (A and B) E. chaffeensis ( Ech ) -infected RF/6A cells were transfected with plasmids encoding 2×FYVE-GFP or FLAG-PIK3C3/VPS34. At 15 h p.t. (2 d p.i.), cells were fixed and stained with DAPI to indicate E. chaffeensis (pseudocolored in red). PIK3C3 was labeled with mouse anti-FLAG. Merged/DIC, fluorescence image merged with differential interference contrast (DIC) image. Each boxed area is enlarged 4-fold on the right. N, nucleus; scale bars: 10 μm. (C) The percent colocalization of E. chaffeensis inclusions with PIK3C3 or 2×FYVE was determined by counting 10 to 20 inclusions per cell in 5 to 10 cells per experiment from 3 independent experiments. (D) PtdIns3P levels are increased in E. chaffeensis -infected THP-1 cells. Uninfected or E. chaffeensis -infected THP-1 cells (2 × 10 6 cells) at 1 d p.i. were collected, and PtdIns3P lipids were purified and the amount determined by competitive ELISA. Assays were carried out in triplicate. Data are presented as the mean ± standard deviation. * Significantly different by the Student t test ( P

    Article Snippet: Co-immunoprecipitation Uninfected or E. chaffeensis -infected THP-1 cells at 2 d p.i. were lysed in modified lysis buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 1% NP40 [USB, 19638], 5% glycerol, and 1% protease inhibitor cocktail III [Calbiochem, 539134]) for 15 min and immunoprecipitated for 2 h with rabbit anti-Etf-1, -BECN1, or -PIK3C3, or mouse anti-RAB5 IgG that was cross-linked to protein A/G-magnetic beads (Pierce, 88805).

    Techniques: Infection, Transfection, Staining, Labeling, Fluorescence, Purification, Competitive ELISA, Standard Deviation

    E. chaffeensis infection requires BECN1 and is enhanced by rapamycin. (A) Depletion of BECN1 suppresses E. chaffeensis infection. HEK293 cells were transfected with BECN1 siRNA or control scrambled siRNA (Neg.) for 40 h and then infected with E. chaffeensis for 36 h. Western blotting was performed using anti-P28, ACT/actin, and BECN1. The values under the bands show the relative ratio of band intensities vs. ACT/actin, with the ratios of those from control siRNA set as 1. (B) Spautin-1 inhibits E. chaffeensis growth. E. chaffeensis –infected THP-1 cells were treated at 1 d p.i. with DMSO solvent control or with 1 μM or 10 μM spautin-1 and incubated for an additional 2 d. Infection was assessed at 3 d p.i. by Diff-Quik staining to determine the percent of infected cells. *, Significantly different by the Tukey HSD test ( P

    Journal: Autophagy

    Article Title: Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase

    doi: 10.1080/15548627.2016.1217369

    Figure Lengend Snippet: E. chaffeensis infection requires BECN1 and is enhanced by rapamycin. (A) Depletion of BECN1 suppresses E. chaffeensis infection. HEK293 cells were transfected with BECN1 siRNA or control scrambled siRNA (Neg.) for 40 h and then infected with E. chaffeensis for 36 h. Western blotting was performed using anti-P28, ACT/actin, and BECN1. The values under the bands show the relative ratio of band intensities vs. ACT/actin, with the ratios of those from control siRNA set as 1. (B) Spautin-1 inhibits E. chaffeensis growth. E. chaffeensis –infected THP-1 cells were treated at 1 d p.i. with DMSO solvent control or with 1 μM or 10 μM spautin-1 and incubated for an additional 2 d. Infection was assessed at 3 d p.i. by Diff-Quik staining to determine the percent of infected cells. *, Significantly different by the Tukey HSD test ( P

    Article Snippet: Co-immunoprecipitation Uninfected or E. chaffeensis -infected THP-1 cells at 2 d p.i. were lysed in modified lysis buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 1% NP40 [USB, 19638], 5% glycerol, and 1% protease inhibitor cocktail III [Calbiochem, 539134]) for 15 min and immunoprecipitated for 2 h with rabbit anti-Etf-1, -BECN1, or -PIK3C3, or mouse anti-RAB5 IgG that was cross-linked to protein A/G-magnetic beads (Pierce, 88805).

    Techniques: Infection, Transfection, Western Blot, Activated Clotting Time Assay, Incubation, Diff-Quik, Staining

    NPEPPS-GFP colocalizes with Etf-1 in cotransfected cells, traffics to E. chaffeensis inclusions, and reduces aggregation of Q103-HTT. (A and B) NPEPPS/PSA-GFP colocalizes with Etf-1 in cotransfected cells and surrounds E. chaffeensis inclusions. (A) DH82 cells were sequentially transfected first with Etf-1 and 1 d later with NPEPPS-GFP. At 1 d p.t. with NPEPPS-GFP, cells were immunostained with anti-Etf-1 (AF555). White arrows indicate the colocalization between the 2 proteins. (B) E. chaffeensis -infected RF/6A cells were transfected with NPEPPS-GFP at 1 d p.i. and stained with DAPI at 1 d p.t. (2 d p.i.). Merged/DIC, fluorescence image merged with DIC image. The boxed area is enlarged on the right. Scale bars: 15 μm. (C) PAQ-22 inhibits E. chaffeensis replication in THP-1 cells. E. chaffeensis -infected THP-1 cells were incubated with 0.1% DMSO (control) or 10 or 100 μM PAQ-22. qPCR of E. chaffeensis 16S rDNA normalized to human GAPDH . *, Significantly different by the Tukey HSD test ( P

    Journal: Autophagy

    Article Title: Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase

    doi: 10.1080/15548627.2016.1217369

    Figure Lengend Snippet: NPEPPS-GFP colocalizes with Etf-1 in cotransfected cells, traffics to E. chaffeensis inclusions, and reduces aggregation of Q103-HTT. (A and B) NPEPPS/PSA-GFP colocalizes with Etf-1 in cotransfected cells and surrounds E. chaffeensis inclusions. (A) DH82 cells were sequentially transfected first with Etf-1 and 1 d later with NPEPPS-GFP. At 1 d p.t. with NPEPPS-GFP, cells were immunostained with anti-Etf-1 (AF555). White arrows indicate the colocalization between the 2 proteins. (B) E. chaffeensis -infected RF/6A cells were transfected with NPEPPS-GFP at 1 d p.i. and stained with DAPI at 1 d p.t. (2 d p.i.). Merged/DIC, fluorescence image merged with DIC image. The boxed area is enlarged on the right. Scale bars: 15 μm. (C) PAQ-22 inhibits E. chaffeensis replication in THP-1 cells. E. chaffeensis -infected THP-1 cells were incubated with 0.1% DMSO (control) or 10 or 100 μM PAQ-22. qPCR of E. chaffeensis 16S rDNA normalized to human GAPDH . *, Significantly different by the Tukey HSD test ( P

    Article Snippet: Co-immunoprecipitation Uninfected or E. chaffeensis -infected THP-1 cells at 2 d p.i. were lysed in modified lysis buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 1% NP40 [USB, 19638], 5% glycerol, and 1% protease inhibitor cocktail III [Calbiochem, 539134]) for 15 min and immunoprecipitated for 2 h with rabbit anti-Etf-1, -BECN1, or -PIK3C3, or mouse anti-RAB5 IgG that was cross-linked to protein A/G-magnetic beads (Pierce, 88805).

    Techniques: Transfection, Infection, Staining, Fluorescence, Incubation, Real-time Polymerase Chain Reaction

    Host preincorporated amino acids are taken up by E. chaffeensis ( Ech ) in a host class III PtdIns3K-dependent manner. (A) THP-1 cells were prelabeled with [ 3 H]glutamine for 1 d and then infected with E. chaffeensis for 2 d in the absence of [ 3 H]glutamine. Infected cells were treated with 2 mM 3-MA or solvent control for an additional 6 h. E. chaffeensis was purified from infected cells, and the [ 3 H]glutamine incorporated in E. chaffeensis was determined by liquid scintillation counter and normalized to the total protein amount. Data are presented as the mean ± standard deviation of triplicate samples. *, Significantly different ( P

    Journal: Autophagy

    Article Title: Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase

    doi: 10.1080/15548627.2016.1217369

    Figure Lengend Snippet: Host preincorporated amino acids are taken up by E. chaffeensis ( Ech ) in a host class III PtdIns3K-dependent manner. (A) THP-1 cells were prelabeled with [ 3 H]glutamine for 1 d and then infected with E. chaffeensis for 2 d in the absence of [ 3 H]glutamine. Infected cells were treated with 2 mM 3-MA or solvent control for an additional 6 h. E. chaffeensis was purified from infected cells, and the [ 3 H]glutamine incorporated in E. chaffeensis was determined by liquid scintillation counter and normalized to the total protein amount. Data are presented as the mean ± standard deviation of triplicate samples. *, Significantly different ( P

    Article Snippet: Co-immunoprecipitation Uninfected or E. chaffeensis -infected THP-1 cells at 2 d p.i. were lysed in modified lysis buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 1% NP40 [USB, 19638], 5% glycerol, and 1% protease inhibitor cocktail III [Calbiochem, 539134]) for 15 min and immunoprecipitated for 2 h with rabbit anti-Etf-1, -BECN1, or -PIK3C3, or mouse anti-RAB5 IgG that was cross-linked to protein A/G-magnetic beads (Pierce, 88805).

    Techniques: Infection, Purification, Standard Deviation