Journal: bioRxiv

Article Title: Structural analysis of the master regulator Rns reveals a small molecule inhibitor of enterotoxigenic Escherichia coli virulence

doi: 10.1101/2020.10.05.326769

Figure Lengend Snippet: A . Structure of SeMet-Rns showing decanoic acid modeled in the ligand binding pocket with α-helices in blue, β-sheets in orange, coils in gray, and the decanoic acid in pink. The N-terminal and DNA binding domains are labeled. B . Proposed biological dimer with the N-terminal domains of the two monomers in orange or blue and both DNA binding domains in teal. The N-terminal α-helices are labeled.

Article Snippet: The sequence encoding Rns was optimized to remove rare codons and flanking sequences to insert into the plasmid pCDB24, a gift from Dr. Bahl (Institute for Protein Innovation), which contains a N-terminal 10xHis-SUMO tag (pCDB24 addgene.org), were added.

Techniques: Ligand Binding Assay, Binding Assay, Labeling

Journal: bioRxiv

Article Title: Structural analysis of the master regulator Rns reveals a small molecule inhibitor of enterotoxigenic Escherichia coli virulence

doi: 10.1101/2020.10.05.326769

Figure Lengend Snippet: A . Superposition of the native (orange) and SeMet-Rns (blue) structures, the two domains were aligned using the long helix in the DNA binding domain (Phe205-Glu223), showing how the N-terminal domain is shifted away from the DBD in the SeMet structure. B . Detail showing the β-sheet Is shifted by ∼2 Å from the DBD in the SeMet structure compared to the native structure. C . Surface representation of the SeMet-Rns structure in blue with the decanoic acid in pink showing how the fatty acid is exposed to solvent. D . Surface representation of the native-Rns in orange with the decanoic acid in purple showing how the ligand is enclosed in more of a pocket.

Article Snippet: The sequence encoding Rns was optimized to remove rare codons and flanking sequences to insert into the plasmid pCDB24, a gift from Dr. Bahl (Institute for Protein Innovation), which contains a N-terminal 10xHis-SUMO tag (pCDB24 addgene.org), were added.

Techniques: Binding Assay

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The p90rsk-mediated signaling of ethanol-induced cell proliferation in HepG2 cell line

doi: 10.4196/kjpp.2016.20.6.595

Figure Lengend Snippet: Serum-starved HepG2 cells were incubated with ethanol for 24 hours at the indicated concentration. The concentration of phosphorylated-p90rsk in the cell lysate was determined by ELISA. Data are expressed as means±S.E of four experiments (student's t -test; * p<0.05 vs. control).

Article Snippet: Aprotinin, leupeptin, bovine serum albumin (BSA), β-mercaptoethanol, ethylene glycol-bis-(β-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), phenylmethyl-sulfonylfluoride (PMSF), thiazolyl blue tetrazolium bromide, ethylenediamine tetra acetic acid (EDTA), Hank's Balanced Salt Solution-Modified (HBSS), Phosphatase Inhibitor Cocktail 3 and PD98059 were purchased from Sigma Chemical Co. (St. Louis, MO, USA); Fetal bovine serum (FBS), Antibiotic-Antimycotic (penicillin, streptomycin, amphotericin B), TRIzol reagent and trypsin-EDTA from Invitrogen (Grand Island, NY, USA); Ethanol from Merck (Darmstadt, Germany) Tris-buffered Saline(TBS), Dulbecco's modified Eagle's medium (DMEM) and phosphatebuffered saline (PBS) from Welgene Inc. (Daegu, South Korea); p90rsk ELISA kit from Cell Signaling Technology (Beverly, MA, USA); Actin antibody SL-0101 and cariporide and ERK1 antibody from Santa Cruz Biotechnology (Santa Cruz, CA, USA) goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP from Bethyl (Montgomery, TX, USA); NHE1 antibody from Abcam (Cambridge, UK); Raddinbow prestained molecular weight marker from Amersham (Arlington Heights, IL, USA); Ammonium persulfate from PerkinElmer Life Sciences (Boston, MA, USA); nitrocellulose (NC) membrane, Tris/Glycine/SDS buffer, Tris/Glycine buffer, Restore TM Western Blot Stripping Buffer from Thermo (Rockford, IL, USA); DEPC-DW from BIONEER (Daejeon, South Korea); TOPscript™ cDNA Synthesis kit and TOPreal™ qPCR 2X PreMIX (SYBR Green with low ROX) from Enzynomics (Daejeon, South Korea); 30% acrylamide/bis solution from BioRad (Richmond, CA, USA).

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The p90rsk-mediated signaling of ethanol-induced cell proliferation in HepG2 cell line

doi: 10.4196/kjpp.2016.20.6.595

Figure Lengend Snippet: Serum-starved HepG2 cells were incubated with ethanol for 24 hours with or without PD98059 (ERK inhibitor) at the indicated concentration. The concentration of phosphorylated-p90rsk in the cell lysate was determined by ELISA. Data are expressed as means±S. E of four experiments (student's t -test; * p<0.05 vs. control, # p<0.05 vs. ethanol alone).

Article Snippet: Aprotinin, leupeptin, bovine serum albumin (BSA), β-mercaptoethanol, ethylene glycol-bis-(β-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), phenylmethyl-sulfonylfluoride (PMSF), thiazolyl blue tetrazolium bromide, ethylenediamine tetra acetic acid (EDTA), Hank's Balanced Salt Solution-Modified (HBSS), Phosphatase Inhibitor Cocktail 3 and PD98059 were purchased from Sigma Chemical Co. (St. Louis, MO, USA); Fetal bovine serum (FBS), Antibiotic-Antimycotic (penicillin, streptomycin, amphotericin B), TRIzol reagent and trypsin-EDTA from Invitrogen (Grand Island, NY, USA); Ethanol from Merck (Darmstadt, Germany) Tris-buffered Saline(TBS), Dulbecco's modified Eagle's medium (DMEM) and phosphatebuffered saline (PBS) from Welgene Inc. (Daegu, South Korea); p90rsk ELISA kit from Cell Signaling Technology (Beverly, MA, USA); Actin antibody SL-0101 and cariporide and ERK1 antibody from Santa Cruz Biotechnology (Santa Cruz, CA, USA) goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP from Bethyl (Montgomery, TX, USA); NHE1 antibody from Abcam (Cambridge, UK); Raddinbow prestained molecular weight marker from Amersham (Arlington Heights, IL, USA); Ammonium persulfate from PerkinElmer Life Sciences (Boston, MA, USA); nitrocellulose (NC) membrane, Tris/Glycine/SDS buffer, Tris/Glycine buffer, Restore TM Western Blot Stripping Buffer from Thermo (Rockford, IL, USA); DEPC-DW from BIONEER (Daejeon, South Korea); TOPscript™ cDNA Synthesis kit and TOPreal™ qPCR 2X PreMIX (SYBR Green with low ROX) from Enzynomics (Daejeon, South Korea); 30% acrylamide/bis solution from BioRad (Richmond, CA, USA).

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The p90rsk-mediated signaling of ethanol-induced cell proliferation in HepG2 cell line

doi: 10.4196/kjpp.2016.20.6.595

Figure Lengend Snippet: Serum-starved HepG2 cells were treated for 24 hours with ethanol (20 mM) alone or in combination with SL0101 (p90rsk inhibitor) at each concentration. Cell viability was assessed with MTT assay. Data are expressed as means±S.E of three experiments (student's t -test; * p<0.05 vs. normal group, # p<0.05 vs. ethanol alone).

Article Snippet: Aprotinin, leupeptin, bovine serum albumin (BSA), β-mercaptoethanol, ethylene glycol-bis-(β-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), phenylmethyl-sulfonylfluoride (PMSF), thiazolyl blue tetrazolium bromide, ethylenediamine tetra acetic acid (EDTA), Hank's Balanced Salt Solution-Modified (HBSS), Phosphatase Inhibitor Cocktail 3 and PD98059 were purchased from Sigma Chemical Co. (St. Louis, MO, USA); Fetal bovine serum (FBS), Antibiotic-Antimycotic (penicillin, streptomycin, amphotericin B), TRIzol reagent and trypsin-EDTA from Invitrogen (Grand Island, NY, USA); Ethanol from Merck (Darmstadt, Germany) Tris-buffered Saline(TBS), Dulbecco's modified Eagle's medium (DMEM) and phosphatebuffered saline (PBS) from Welgene Inc. (Daegu, South Korea); p90rsk ELISA kit from Cell Signaling Technology (Beverly, MA, USA); Actin antibody SL-0101 and cariporide and ERK1 antibody from Santa Cruz Biotechnology (Santa Cruz, CA, USA) goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP from Bethyl (Montgomery, TX, USA); NHE1 antibody from Abcam (Cambridge, UK); Raddinbow prestained molecular weight marker from Amersham (Arlington Heights, IL, USA); Ammonium persulfate from PerkinElmer Life Sciences (Boston, MA, USA); nitrocellulose (NC) membrane, Tris/Glycine/SDS buffer, Tris/Glycine buffer, Restore TM Western Blot Stripping Buffer from Thermo (Rockford, IL, USA); DEPC-DW from BIONEER (Daejeon, South Korea); TOPscript™ cDNA Synthesis kit and TOPreal™ qPCR 2X PreMIX (SYBR Green with low ROX) from Enzynomics (Daejeon, South Korea); 30% acrylamide/bis solution from BioRad (Richmond, CA, USA).

Techniques: Concentration Assay, MTT Assay

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The p90rsk-mediated signaling of ethanol-induced cell proliferation in HepG2 cell line

doi: 10.4196/kjpp.2016.20.6.595

Figure Lengend Snippet: A, Serum-starved HepG2 cells were treated for 24 hours with ethanol (20 mM) alone or in combination with SL0101 (p90rsk inhibitor) at each concentration. Identical amounts of lysate proteins were subjected to 7.5% SDS-PAGE and immunoblotted (IB) with anti-NHE1 antibody. β–actin content within lystates is shown as an loading control. B, the graph represents fold expression of NHE1 relative to β–actin averaged from three independent experiments. Data are expressed as means±S.E of four experiments (student's t -test; * p<0.05 vs. control, # p<0.05 vs. ethanol alone).

Article Snippet: Aprotinin, leupeptin, bovine serum albumin (BSA), β-mercaptoethanol, ethylene glycol-bis-(β-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), phenylmethyl-sulfonylfluoride (PMSF), thiazolyl blue tetrazolium bromide, ethylenediamine tetra acetic acid (EDTA), Hank's Balanced Salt Solution-Modified (HBSS), Phosphatase Inhibitor Cocktail 3 and PD98059 were purchased from Sigma Chemical Co. (St. Louis, MO, USA); Fetal bovine serum (FBS), Antibiotic-Antimycotic (penicillin, streptomycin, amphotericin B), TRIzol reagent and trypsin-EDTA from Invitrogen (Grand Island, NY, USA); Ethanol from Merck (Darmstadt, Germany) Tris-buffered Saline(TBS), Dulbecco's modified Eagle's medium (DMEM) and phosphatebuffered saline (PBS) from Welgene Inc. (Daegu, South Korea); p90rsk ELISA kit from Cell Signaling Technology (Beverly, MA, USA); Actin antibody SL-0101 and cariporide and ERK1 antibody from Santa Cruz Biotechnology (Santa Cruz, CA, USA) goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP from Bethyl (Montgomery, TX, USA); NHE1 antibody from Abcam (Cambridge, UK); Raddinbow prestained molecular weight marker from Amersham (Arlington Heights, IL, USA); Ammonium persulfate from PerkinElmer Life Sciences (Boston, MA, USA); nitrocellulose (NC) membrane, Tris/Glycine/SDS buffer, Tris/Glycine buffer, Restore TM Western Blot Stripping Buffer from Thermo (Rockford, IL, USA); DEPC-DW from BIONEER (Daejeon, South Korea); TOPscript™ cDNA Synthesis kit and TOPreal™ qPCR 2X PreMIX (SYBR Green with low ROX) from Enzynomics (Daejeon, South Korea); 30% acrylamide/bis solution from BioRad (Richmond, CA, USA).

Techniques: Concentration Assay, SDS Page, Expressing

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The p90rsk-mediated signaling of ethanol-induced cell proliferation in HepG2 cell line

doi: 10.4196/kjpp.2016.20.6.595

Figure Lengend Snippet: HepG2 cells were treated with 20 mM of ethanol alone or in combination of p90rsk or NHE1 inhibitor for 24 hours. Total RNA was extracted and subjected to quantitative RT-PCR (qRT-PCR) for Bcl-2 mRNA. Data are expressed as means±S.E of three experiments (student's t -test; * p<0.05 vs. control, # p<0.05 vs. ethanol alone).

Article Snippet: Aprotinin, leupeptin, bovine serum albumin (BSA), β-mercaptoethanol, ethylene glycol-bis-(β-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), phenylmethyl-sulfonylfluoride (PMSF), thiazolyl blue tetrazolium bromide, ethylenediamine tetra acetic acid (EDTA), Hank's Balanced Salt Solution-Modified (HBSS), Phosphatase Inhibitor Cocktail 3 and PD98059 were purchased from Sigma Chemical Co. (St. Louis, MO, USA); Fetal bovine serum (FBS), Antibiotic-Antimycotic (penicillin, streptomycin, amphotericin B), TRIzol reagent and trypsin-EDTA from Invitrogen (Grand Island, NY, USA); Ethanol from Merck (Darmstadt, Germany) Tris-buffered Saline(TBS), Dulbecco's modified Eagle's medium (DMEM) and phosphatebuffered saline (PBS) from Welgene Inc. (Daegu, South Korea); p90rsk ELISA kit from Cell Signaling Technology (Beverly, MA, USA); Actin antibody SL-0101 and cariporide and ERK1 antibody from Santa Cruz Biotechnology (Santa Cruz, CA, USA) goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP from Bethyl (Montgomery, TX, USA); NHE1 antibody from Abcam (Cambridge, UK); Raddinbow prestained molecular weight marker from Amersham (Arlington Heights, IL, USA); Ammonium persulfate from PerkinElmer Life Sciences (Boston, MA, USA); nitrocellulose (NC) membrane, Tris/Glycine/SDS buffer, Tris/Glycine buffer, Restore TM Western Blot Stripping Buffer from Thermo (Rockford, IL, USA); DEPC-DW from BIONEER (Daejeon, South Korea); TOPscript™ cDNA Synthesis kit and TOPreal™ qPCR 2X PreMIX (SYBR Green with low ROX) from Enzynomics (Daejeon, South Korea); 30% acrylamide/bis solution from BioRad (Richmond, CA, USA).

Techniques: Quantitative RT-PCR