pdeltacmv vash1 c168a catalytically dead mutant mscarlet p2a svbp  (Addgene inc)

Journal: The Journal of Biological Chemistry

Article Title: Neuraminidase 1–mediated desialylation of the mucin 1 ectodomain releases a decoy receptor that protects against Pseudomonas aeruginosa lung infection

doi: 10.1074/jbc.RA118.006022

Figure Lengend Snippet: Purified Pa flagellin recapitulates Pa-provoked MUC1-ED shedding in vivo. A, BALB/c mice were administered i.n. with 10 ng/mouse of PAK flagellin or the PBS vehicle. At increasing times posttreatment, MUC1-ED levels in BALF were quantified and normalized to BALF protein. B, mice were administered increasing doses of PAK flagellin. At 24 h posttreatment, MUC1-ED levels in BALF were quantified and normalized to BALF protein. C, lysates of PAK and PAO1 were processed for flagellin immunoblotting using mouse anti-FlaA (lane 1) or anti-FlaB (lane 2) polyclonal antisera. Molecular masses in kDa are indicated on the left. D and E, PAK (D) or PAO1 (E) (1.0 × 107 CFUs) were incubated for 30 min at 4 °C with undiluted or decreasing dilutions of 100 μl of anti-FlaA (D) or anti-FlaB (E) antisera or the PBS control. The bacteria were washed and processed for adhesion to Ad-NEU1–infected (multiplicity of infection (m.o.i.) = 100) A549 cells. F, PAK (1.0 × 105 CFU) was preincubated with 10 μl of anti-FlaA antiserum or nonimmune mouse serum, and PAO1 (1.0 × 105 CFUs) was preincubated with 10 μl of anti-FlaB antiserum or nonimmune mouse serum for 30 min at 4 °C and washed. BALB/c mice were infected i.n. with the bacteria or administered the PBS vehicle. At 24 h postinfection, MUC1-ED levels in BALF were quantified and normalized to BALF protein. Error bars represent the mean ± S.E. values (n = 3). *, increased MUC1-ED levels compared with (A and B) PBS controls or (F) nonimmune mouse serum at p < 0.05. **, (E) decreased Pa adhesion compared with PBS controls, or (F) decreased MUC1-ED levels compared with nonimmune Ab, at p < 0.05. The results are representative of three independent experiments.

Article Snippet: Adenoviral constructs Recombinant Ad encoding FLAG-tagged human NEU1 (Ad-NEU1), Ad encoding the catalytically dead NEU1 mutant, Ad-NEU1-G68V, Ad-GFP, and Ad-Null were generated using the AdEasy Adenoviral Vector System (Stratagene, La Jolla, CA) as described ( 19 , 20 , 57 , 59 ).

Techniques: Purification, In Vivo, Western Blot, Incubation, Infection

Journal: The Journal of Biological Chemistry

Article Title: Neuraminidase 1–mediated desialylation of the mucin 1 ectodomain releases a decoy receptor that protects against Pseudomonas aeruginosa lung infection

doi: 10.1074/jbc.RA118.006022

Figure Lengend Snippet: Pa lung infection increases NEU1-MUC1 and PPCA-MUC1 association and MUC1-ED desialylation. A–E and G–J, BALB/c mice were administered i.n. with 1.0 × 105 CFUs/mouse of PAK or the PBS vehicle. At 24 h postchallenge, BALF and lung tissues were collected, and lungs were homogenized. A, lung homogenates were immunoprecipitated with anti-MUC1-CD (lanes 1, 2, 5, 6), anti-NEU1 (lanes 3, 4), or anti-PPCA (lanes 7, 8) Abs. The MUC1-CD immunoprecipitates were processed for NEU1 (lanes 1, 2) or PPCA (lanes 5, 6) immunoblotting, and the NEU1 (lanes 3, 4) and PPCA (lanes 7, 8) immunoprecipitates were processed for MUC1-CD immunoblotting (upper panels). To control for protein loading and transfer, the immunoblots were stripped and reprobed with the immunoprecipitating Ab (lower panels). B–E, densitometric analyses of the blots in (A). Error bars represent mean ± S.E. MUC1-CD, NEU1, or PPCA signal normalized to the immunoprecipitate signal in the same lane on the same stripped and reprobed blot (n = 3). *, increased normalized lung NEU1, MUC1-CD, or PPCA signal compared with PBS controls at p < 0.05. F, to validate PNA selectivity, the negative control, fetuin (lane 1), and the positive control, asialofetuin (lane 2), 1.0 μg each, were processed for PNA lectin blotting. G, BALF (lanes 1 and 2) and lung homogenates (lanes 3 and 4) from mice administered i.n. with PAK or the PBS vehicle were processed for MUC1-ED immunoblotting. H, BALF (lanes 1 and 2) and lung homogenates (lanes 3 and 4) were incubated with PNA-agarose and the PNA-binding proteins processed for MUC1-ED immunoblotting. I and J, densitometric analyses of the blots in (H). Error bars represent mean ± S.E. BALF/lung desialylated MUC1-ED signal (n = 3). *, increased BALF MUC1-ED signal compared with PBS controls at p < 0.05. (A and F–H), molecular masses in kDa are indicated on the left. IP, immunoprecipitate; IB, immunoblot; IB*, immunoblot after stripping. PD, pulldown. The results are representative of three independent experiments.

Article Snippet: Adenoviral constructs Recombinant Ad encoding FLAG-tagged human NEU1 (Ad-NEU1), Ad encoding the catalytically dead NEU1 mutant, Ad-NEU1-G68V, Ad-GFP, and Ad-Null were generated using the AdEasy Adenoviral Vector System (Stratagene, La Jolla, CA) as described ( 19 , 20 , 57 , 59 ).

Techniques: Infection, Immunoprecipitation, Western Blot, Negative Control, Positive Control, Incubation, Binding Assay, Stripping Membranes

Journal: The Journal of Biological Chemistry

Article Title: Neuraminidase 1–mediated desialylation of the mucin 1 ectodomain releases a decoy receptor that protects against Pseudomonas aeruginosa lung infection

doi: 10.1074/jbc.RA118.006022

Figure Lengend Snippet: NEU1 is required for maximal Pa-induced MUC1-ED desialylation and shedding. A–D, BALB/c mice were administered intraperitoneally with the indicated amounts of C9-BA-DANA, DANA, or the PBS vehicle. At 24 h postadministration, mice were infected i.n. with 1.0 × 105 CFUs/mouse of PAK. At 24 h postinfection, BALF was collected. A, BALFs were incubated with PNA-agarose and the PNA-binding proteins processed for MUC1-ED immunoblotting. B, densitometric analyses of the blots in (A). B and C, error bars represent mean ± S.E. BALF desialylated MUC1-ED signal or shed MUC1-ED levels (n = 3). C, BALF MUC1-ED levels were quantified by ELISA and normalized to BALF protein. D, BALFs were processed for MUC1-ED immunoblotting. **, decreased desialylated MUC1-ED signal or MUC1-ED level compared with PBS control at p < 0.05. †, decreased desialylated MUC1-ED signal or MUC1-ED level in mice administered DANA compared with mice administered C9-BA-DANA at p < 0.05. E–G, BALB/c mice were administered i.t. with Ad-NEU1, Ad-NEU1-G68V, or Ad-Null. At 3 days postinfection, BALF was collected. E, BALFs were incubated with PNA-agarose and the PNA-binding proteins processed for MUC1-ED immunoblotting. F, densitometric analyses of the blots in (E). G, BALF MUC1-ED levels were quantified by ELISA and normalized to BALF protein. F and G, error bars represent mean ± S.E. BALF desialylated MUC1-ED signal or MUC1-ED levels (n = 5). *, increased desialylated MUC1-ED signal or MUC1-ED level compared with Ad-Null or Ad-NEU1-G68V controls at p < 0.05. A, D, and E, Molecular masses in kDa are indicated on the left. H, BALB/c mice were infected i.n. with 1.0 × 105 CFUs of WT NanPs-expressing PAO1 or the NanPs-deficient PAO1/NanPs− isogenic mutant. At 24 h postinfection, MUC1-ED levels in BALF were quantified and normalized to BALF protein (n = 6). I, HEK293 cells were transfected with plasmids encoding for WT MUC1 or the MUC1-ED S317A protease recognition site mutant and incubated for 24 h. The ECs were infected with increasing m.o.i.s of Ad-GFP or Ad-NEU1. After 48 h, shed MUC1-ED levels in cell culture supernatants were quantified by ELISA and normalized to total supernatant protein. Error bars represent mean ± S.E. shed MUC1-ED levels (n = 3). *, increased MUC1-ED levels versus Ad-GFP-infected MUC1-HEK293 cells at p < 0.05. **, decreased MUC1-ED levels versus Ad-NEU1-infected MUC1-HEK293 cells at p < 0.05. The results are representative of three independent experiments. IB, immunoblot; PD, pulldown.

Article Snippet: Adenoviral constructs Recombinant Ad encoding FLAG-tagged human NEU1 (Ad-NEU1), Ad encoding the catalytically dead NEU1 mutant, Ad-NEU1-G68V, Ad-GFP, and Ad-Null were generated using the AdEasy Adenoviral Vector System (Stratagene, La Jolla, CA) as described ( 19 , 20 , 57 , 59 ).

Techniques: Infection, Incubation, Binding Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Mutagenesis, Transfection, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Neuraminidase 1–mediated desialylation of the mucin 1 ectodomain releases a decoy receptor that protects against Pseudomonas aeruginosa lung infection

doi: 10.1074/jbc.RA118.006022

Figure Lengend Snippet: MUC1-ED–enriched BALF inhibits flagellin binding and Pa adhesion to airway ECs and Pa motility. BALB/c mice were administered i.n. with 1.0 × 105 CFUs/mouse of PAK or the PBS vehicle. At increasing times postadministration, BALF was collected. A and B, A549 cells were infected with Ad-NEU1 (m.o.i. = 100) and cultured for 48 h. A, Alexa Fluor 594–labeled flagellin was incubated for 30 min at 4 °C with BALF from uninfected or PAK-infected mice and assayed for binding to Ad-NEU1–infected A549 cells. B, PAK was incubated for 30 min at 4 °C with BALF from uninfected or PAK-infected mice and assayed for adhesion to Ad-NEU1–infected A549 cells. C, PAK was incubated for 30 min at 4 °C with BALF from uninfected or PAK-infected mice and assayed for bacterial motility. A–C, error bars represent mean ± S.E. flagellin binding or Pa adhesion or motility (n = 3). D, BALFs incubated with anti-MUC1-ED or control Abs were processed for MUC1-ED immunoblotting. To control for equal protein and loading, the blot was stripped and reprobed for β-tubulin. Molecular masses in kDa are indicated on the left. IB, immunoblot; IB*, immunoblot after stripping. A–C, in selected experiments, BALF from PAK-infected mice collected at 48 h postinfection was incubated with anti-MUC1-ED Ab or a species- and isotype-matched nonimmune IgG control. Igs were immobilized on protein G–agarose and removed by centrifugation. The MUC1-ED–immunodepleted or control IgG-treated BALFs were incubated with Alexa Fluor 594–flagellin or Pa, and processed for (A) flagellin binding or (B) bacterial adhesion to Ad-NEU1–infected A549 cells or (C) bacterial motility. *, increased flagellin binding, or Pa adhesion or motility, following incubation with MUC1-ED–immunodepleted BALFs compared with nonimmune IgG-treated BALFs at p < 0.05. **, decreased flagellin binding or Pa adhesion or motility following incubation with BALFs from Pa-infected mice compared with incubation with BALF from uninfected controls at p < 0.05. The results are representative of three independent experiments.

Article Snippet: Adenoviral constructs Recombinant Ad encoding FLAG-tagged human NEU1 (Ad-NEU1), Ad encoding the catalytically dead NEU1 mutant, Ad-NEU1-G68V, Ad-GFP, and Ad-Null were generated using the AdEasy Adenoviral Vector System (Stratagene, La Jolla, CA) as described ( 19 , 20 , 57 , 59 ).

Techniques: Binding Assay, Infection, Cell Culture, Labeling, Incubation, Western Blot, Stripping Membranes, Centrifugation

Journal: The Journal of Biological Chemistry

Article Title: Neuraminidase 1–mediated desialylation of the mucin 1 ectodomain releases a decoy receptor that protects against Pseudomonas aeruginosa lung infection

doi: 10.1074/jbc.RA118.006022

Figure Lengend Snippet: Deglycosylated protein backbone of human MUC1-ED inhibits Pa adhesion to human airway ECs. A, PAK was incubated with human MUC1-ED isolated from supernatants of A549 airway ECs infected with Ad-NEU1, Ad-NEU1–G68V, or Ad-GFP (m.o.i. = 100), washed, and assayed for adhesion to fresh, unmanipulated A549 cells (n = 3). B, Alexa Fluor 594–labeled PAK flagellin was incubated for 30 min at 37 °C with 25 mm of Gal or GalNAc, 25 mm Gal plus 25 mm GalNAc, or the PBS vehicle control. The ECs were washed and processed for flagellin binding by fluorometry (n = 3). C, PAK was incubated with human MUC1-ED isolated from the supernatants of A549 cells cultured in the presence of 1.0 μg/ml tunicamycin, 5.0 μm GalNAc-O-bn, or the PBS vehicle control, washed, and assayed for adhesion to fresh, unmanipulated A549 cells (n = 3). D, PAK was incubated with human rMUC1-ED prepared from E. coli transformed with a MUC1 expression plasmid or the empty vector control, washed, and assayed for adhesion to A549 cells (n = 3). Error bars represent mean ± S.E. Pa adhesion (A, C, D) or flagellin binding (B). **, significantly decreased Pa adhesion versus Ad-GFP, PBS, or empty vector controls at p < 0.05. E, human MUC1-ED isolated from A549 cells cultured in the presence of 1.0 μg/ml tunicamycin, 5.0 μm GalNAc-O-bn, or the PBS vehicle control, and E. coli-expressed human rMUC1-ED were processed for MUC1-ED immunoblotting. Molecular masses in kDa are indicated on the left. IB, immunoblot. The results are representative of three independent experiments.

Article Snippet: Adenoviral constructs Recombinant Ad encoding FLAG-tagged human NEU1 (Ad-NEU1), Ad encoding the catalytically dead NEU1 mutant, Ad-NEU1-G68V, Ad-GFP, and Ad-Null were generated using the AdEasy Adenoviral Vector System (Stratagene, La Jolla, CA) as described ( 19 , 20 , 57 , 59 ).

Techniques: Incubation, Isolation, Infection, Labeling, Binding Assay, Cell Culture, Transformation Assay, Expressing, Plasmid Preparation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Neuraminidase 1–mediated desialylation of the mucin 1 ectodomain releases a decoy receptor that protects against Pseudomonas aeruginosa lung infection

doi: 10.1074/jbc.RA118.006022

Figure Lengend Snippet: Human rMUC1-ED inhibits flagellin binding and Pa adhesion to human airway ECs and Pa motility. A, PAK was incubated with 25 μm of human rMUC1-ED for 1 h at 4 °C. The bacteria were incubated with mouse anti-MUC1-ED Ab (panel i) or nonimmune mouse IgG (panel ii), each at 1:5000 dilution, for 1 h at 4 °C, followed by gold-labeled goat anti-mouse IgG secondary Ab at 1:10,000 dilution for 1 h at 4 °C, and examined by transmission immunoelectron microscopy. Each section was photographed at 11,000× or 44,000× (insert). Arrowheads indicate immunogold labeling. Scale bar, 100 nm. B and C, Human rMUC1-ED at the indicated concentrations or the PBS vehicle control were incubated with (B) Alexa Fluor 594–labeled Pa flagellin (n = 3) or (C) WT PAK or the flagellin-deficient PAK/fliC− mutant (n = 3) and assayed for (B) flagellin binding or (C) Pa adhesion to Ad-NEU1–infected A549 cells. D, the indicated concentrations of human rMUC1-ED or the PBS vehicle control were incubated with WT PAK or the PAK/fliC− mutant, and the bacteria were stab-inoculated into 0.3% agar plates and incubated overnight. Pa colony diameter was measured as an indicator of bacterial motility (n = 3). Error bars represent mean ± S.E. flagellin binding (B), Pa adhesion (C), or Pa motility (D). **, significantly decreased flagellin binding or Pa adhesion or motility versus the PBS control at p < 0.05. The results are representative of three independent experiments.

Article Snippet: Adenoviral constructs Recombinant Ad encoding FLAG-tagged human NEU1 (Ad-NEU1), Ad encoding the catalytically dead NEU1 mutant, Ad-NEU1-G68V, Ad-GFP, and Ad-Null were generated using the AdEasy Adenoviral Vector System (Stratagene, La Jolla, CA) as described ( 19 , 20 , 57 , 59 ).

Techniques: Binding Assay, Incubation, Labeling, Transmission Assay, Immuno-Electron Microscopy, Mutagenesis, Infection

Journal: Scientific Reports

Article Title: Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli

doi: 10.1038/srep13477

Figure Lengend Snippet: ( A ) Sequence of the designed sgRNA template. sgRNA targets the non-template DNA strand of the gene-coding region. Base-pairing nucleotides (20 bp) are shown in orange. The dCas9-binding hairpin is in blue. The PAM sequence is shown in red. The Trc promoter is shown in grey. ( B ) This CRISPRi system consists of an inducible dCas9 protein and a designed sgRNA chimera. The dCas9 mutant gene contains two silencing mutations of the RuvC1 and HNH nuclease domains. The sgRNA chimera contains four functional domains: a Trc -inducible promoter, a 20-nucleotide (nt) complementary region for specific DNA binding, a 42-nt dCas9-binding hairpin and a 40-nt transcription terminator derived from S. pyogenes .

Article Snippet: A catalytically dead Cas9 mutant (dCas9) was codon-optimized and synthesized by GenScript (Nanjing, China) (see for the dCas9 sequence). pACYC-dCas9 was constructed by digesting dCas9 from pUC57-dCas9 (GenScript, Nanjing, China) into Nco I/ Hin dIII sites of pACYCDuet-1.

Techniques: Sequencing, Binding Assay, Mutagenesis, Functional Assay, Derivative Assay

Journal: Scientific Reports

Article Title: Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli

doi: 10.1038/srep13477

Figure Lengend Snippet: Control strains contained the (2S)-naringenin heterologous pathway without an RNA-guided dCas9:sgRNA system. The sgRNA-expressing plasmids repressing single or multiple genes were further transformed into the control strain to investigate the effects of these systems on (2S)-naringenin production. Final OD 600 values, average specific growth rates and concentrations of p -coumaric acid and (2S)-naringenin were measured from production strains after a total fermentation time of 48 h.

Article Snippet: A catalytically dead Cas9 mutant (dCas9) was codon-optimized and synthesized by GenScript (Nanjing, China) (see for the dCas9 sequence). pACYC-dCas9 was constructed by digesting dCas9 from pUC57-dCas9 (GenScript, Nanjing, China) into Nco I/ Hin dIII sites of pACYCDuet-1.

Techniques: Control, Expressing, Transformation Assay

Journal: Scientific Reports

Article Title: Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli

doi: 10.1038/srep13477

Figure Lengend Snippet: Plasmids used for multiple gene perturbations.

Article Snippet: A catalytically dead Cas9 mutant (dCas9) was codon-optimized and synthesized by GenScript (Nanjing, China) (see for the dCas9 sequence). pACYC-dCas9 was constructed by digesting dCas9 from pUC57-dCas9 (GenScript, Nanjing, China) into Nco I/ Hin dIII sites of pACYCDuet-1.

Techniques: