Journal: Molecular Biology and Evolution

Article Title: Evolutionary Insights into Premetazoan Functions of the Neuronal Protein Homer

doi: 10.1093/molbev/msu178

Figure Lengend Snippet: Conserved biochemical properties and nuclear localization of Homer in the choanoflagellate Salpingoeca rosetta . ( A ) The protein domain organization of Homer is conserved in S. rosetta and metazoan Homer proteins. The protein domain organizations of Homer homologs from Capsaspora owczarzaki , the choanoflagellates S. rosetta and Monosiga brevicollis, and diverse metazoan taxa were mapped onto the consensus phylogeny from ( A ). Yellow boxes highlight the conserved Enabled/VASP homology 1 (EVH1) domain and the CC domains of Homer. Orange triangle indicates SH3 domain in C. owczarzaki Homer protein. Although Homer was found in almost all metazoan (light gray), choanoflagellate (dark gray), and C. owczarzaki genomes analyzed in this study, it was not detected in any other lineage. ( B ) CD spectroscopy of the C-terminal CC domain of bacterially expressed SrHomer (SrHomerCC). The CD spectrum, with two clearly defined minima at 208 and 222 nm, reveals that SrHomerCC is alpha-helical. ( C and D ) Oligomerization of SrHomer in solution. Size-exclusion chromatography (SEC)-MALS data are shown for full-length SrHomer ( C ) and SrHomerΔCC ( D ). Full-length SrHomer exists predominantly as a tetramer (MW: 167 kDa), whereas the deletion of the C-terminal CC domain renders SrHomer dimeric (MW: 67 kDa). The signal from the refractive index detector (dRI; left, Y axis) is shown as solid, black lines. Molecular mass as calculated across the protein elution peak (right, Y axis) is shown as blue lines. ( E–I ) A rosette colony in which the apical flagellum of each cell is oriented outward. The plasma membrane and flagellum of each cell are revealed by staining with antibodies to beta-tubulin ( E , green). ( F ) DAPI staining reveals DNA in the nucleus of each cell. Arrowhead indicates a representative nucleus from one cell. DNA from prey bacteria (indicated with asterisk) also stains with DAPI. ( G ) SrHomer colocalizes with DNA in the nucleus, as revealed in the merge ( H ). ( I ) In S. rosetta , the nucleus is organized with the electron-dense spherical nucleolus (asterisk) in the center surrounded by a shell of nuclear DNA and nucleoplasm, as revealed by TEM of a thin section through a S. rosetta rosette colony. Inset: Nuclear pores from a single nucleus are indicated with arrowheads. ( J ) Staining with antibodies to nuclear pore complexes (green) revealed that SrHomer (red) is contained within the nuclear membrane and distributed throughout the nucleoplasm. The hollow center of the nucleus suggests that SrHomer is either excluded from the nucleolus or that antibodies to SrHomer did not efficiently penetrate the nucleolus. ( K ) Immunogold labeling of S. rosetta cells with an antibody directed against SrHomer. Gold particles are visible as black dots that localize throughout the nucleus. ( L ) Higher magnification of the region of the nucleus contained within the box in ( K ). Homer was seen to localize to both the nucleoplasm and nucleolus. High densities of Homer localization in the nucleus (arrow) were frequently observed. ( M ) Two independent immunogold labeling experiments (Exp. 1 and Exp. 2) show that Homer localizes predominantly to the nucleus (73% and 81%). Nuclear versus cytosolic localization of gold particles was measured from 25 cells per experiment. Scale bars: 5 µm in ( G ), 500 nm in ( I ) and ( J ), 200 nm in ( K ) and ( L ). Species abbreviations as in .

Article Snippet: The following primary antibodies were used: Mouse monoclonal antibody against β-tubulin (E7, 1:400; Developmental Studies Hybridoma Bank) and nuclear pore complexes (1:100, Covance); rabbit polyclonal antibodies against SrHomer (1:200); and rat Flotillin 1 (1:100, Abcam).

Techniques: Circular Dichroism, Size-exclusion Chromatography, Refractive Index, Clinical Proteomics, Membrane, Staining, Bacteria, Labeling

Journal: Molecular Biology and Evolution

Article Title: Evolutionary Insights into Premetazoan Functions of the Neuronal Protein Homer

doi: 10.1093/molbev/msu178

Figure Lengend Snippet: Mass spectrometric identification of Salpingoeca rosetta Homer-binding proteins. ( A ) In a coimmunoprecipitation experiment, diverse proteins were pulled down by SrHomer antibodies, but not by control antibodies. Whole cell lysates from S. rosetta rosette colonies were used for coimmunoprecipitation experiments and the resulting bound proteins analyzed by SDS-PAGE, silver staining, and LC-MS/MS ( supplementary table S2 , Supplementary Material online). ( B ) Select candidate binding partners of SrHomer identified by LC-MS/MS analysis of SrHomer coimmunoprecipitates. The summed number of unique peptides represents the total number of identified peptides from two independent SrHomer IP experiments. No peptides were detected in the control IP experiments for these proteins, suggesting that they are selective binding partners of SrHomer. Previously known Homer-binding partners from metazoans are marked with an asterisk.

Article Snippet: The following primary antibodies were used: Mouse monoclonal antibody against β-tubulin (E7, 1:400; Developmental Studies Hybridoma Bank) and nuclear pore complexes (1:100, Covance); rabbit polyclonal antibodies against SrHomer (1:200); and rat Flotillin 1 (1:100, Abcam).

Techniques: Binding Assay, Control, SDS Page, Silver Staining, Liquid Chromatography with Mass Spectroscopy

Journal: Molecular Biology and Evolution

Article Title: Evolutionary Insights into Premetazoan Functions of the Neuronal Protein Homer

doi: 10.1093/molbev/msu178

Figure Lengend Snippet: SrHomer interacts directly with SrFlotillin 1 in the Salpingoeca rosetta nucleus. ( A ) Salpingoeca rosetta and human Flotillins 1 and 2 contain a conserved N-terminal SPFH-domain and a C-terminal Flotillin domain. ( B ) Purified GST-SrFlotillin 1 (GST-SrFlot1) pulls down SrHomer from S. rosetta cell lysate. Glutathione-sepharose beads bound to GST alone or GST-SrFlotillin 1 were incubated with S. rosetta cell lysate. The input lane represents 2% of the lysate used in the pull-down experiments. The remaining lanes show elutions from the beads separated by SDS-PAGE and visualized with Coomassie Blue (upper panel) or by immunoblotting with SrHomer antibodies (lower panel). ( C ) Purified His-SrHomer binds directly to GST-SrFlotillin 1 but not GST alone. To measure the binding affinity between SrHomer and SrFlotillin 1, increasing concentrations of His-SrHomer were incubated with 2 µM GST-SrFlotillin 1 (lanes 1–6), or with 2 µM GST (lanes 7–12) bound to glutathione-Sepharose beads for 1 h at RT, washed and analyzed on SDS-PAGE (concentrations of His-SrHomer used for the binding assays in lanes 1–6 and lanes 7–12 were 0.1 µM, 0.25 µM, 0.5 µM, 1 µM, 2 µM, and 4 µM, respectively). Saturated binding of SrHomer to SrFlotillin 1 was reached at approximately 2 µM SrHomer. ( D ) Amounts of precipitated His-SrHomer were measured, normalized to GST-SrFlotillin 1, and plotted. The Kd of the reaction was determined by fitting the data to a Hill function assuming 1:1 binding stoichiometry. The apparent binding Kd is approximately 0.96 µM. ( E–L ) SrFlotillin 1 colocalizes with SrHomer in all solitary S. rosetta cells ( E–H ) and in some (indicated with white arrowhead), but not all, cells within S. rosetta rosette colonies ( I–L ). Subcellular localization of beta-tubulin ( E and I , gray), SrHomer ( F and J , green), and SrFlotillin 1 ( G and K , red) in S. rosetta . ( H and L ). The overlay of SrHomer and SrFlotillin 1 shows colocalization of the two proteins, presumably in the nucleus. Scale bar: 2 µm. Sr, S. rosetta .

Article Snippet: The following primary antibodies were used: Mouse monoclonal antibody against β-tubulin (E7, 1:400; Developmental Studies Hybridoma Bank) and nuclear pore complexes (1:100, Covance); rabbit polyclonal antibodies against SrHomer (1:200); and rat Flotillin 1 (1:100, Abcam).

Techniques: Purification, Incubation, SDS Page, Western Blot, Binding Assay

Journal: Molecular Biology and Evolution

Article Title: Evolutionary Insights into Premetazoan Functions of the Neuronal Protein Homer

doi: 10.1093/molbev/msu178

Figure Lengend Snippet: Homer 1 and Flotillin 1 from vertebrates interact directly and colocalize in the nuclei of primary astrocytes. ( A ) Bacterially expressed His-HsHomer 1 binds to GST-HsFlotillin 1 (GST-HsFlot1) but not GST. To measure the binding affinity between HsHomer and HsFlotillin 1, increasing concentrations of His-HsHomer were incubated with 2 µM GST-HsFlotillin 1 (lanes 1–6) or with 2 µM GST (lanes 7–12) bound to glutathione-Sepharose beads for 1 h at RT, washed, and analyzed on SDS-PAGE (concentrations of His-HsHomer 1 used for the binding assays in lanes 1–6 and lanes 7–12 were 0.1 µM, 0.25 µM, 0.5 µM, 1 µM, 2 µM, and 4 µM, respectively). Saturated binding of HsHomer 1 to HsFlotillin 1 was reached at approximately 2 µM SrHomer. ( B ) The Kd of the reaction was determined by fitting the data to a Hill function assuming 1:1 binding stoichiometry. The apparent binding Kd is approximately 0.83 µM. ( C–G ) Homer 1 and Flotillin 1 do not appear to colocalize in rat hippocampal neurons. Hippocampal neurons (12 days in vitro) were stained with DAPI ( C , blue), Flotillin 1 antibodies ( D , green), and Homer 1 antibodies ( E , red). Although Flotillin 1 and Homer 1 both form puncta throughout the dendrites and cytoplasm, both appear to be excluded from the nucleus. An overlay of Flotillin 1 and Homer 1 is shown in panel ( F ). ( G ) Segment of dendrite magnified from ( F ) shows little colocalization between Flotillin 1 and Homer 1. ( H–K ) Homer 1 localizes to the nucleus in rat astrocytes (4 days in vitro) as shown by staining with DAPI ( H , blue), antibodies to the astrocyte marker GFAP (I, green), and antibodies to Homer 1 ( J , red). ( K ) The overlay of DAPI, GFAP, and Homer 1 reveals that Homer 1 colocalizes with DNA in the nucleus. ( L–O ) Flotillin 1 and Homer 1 colocalize in rat astrocyte nuclei. Subcellular localization of DNA ( L , blue), Flotillin 1 ( M , green), and Homer 1 ( N , red) are shown. ( O ) The overlay of DAPI-stained DNA, Flotillin1, and Homer 1 shows colocalization in the nucleus. Scale bars: 10 µm in ( O ) and ( F ), 5 µm in ( G ). Hs , Homo sapiens .

Article Snippet: The following primary antibodies were used: Mouse monoclonal antibody against β-tubulin (E7, 1:400; Developmental Studies Hybridoma Bank) and nuclear pore complexes (1:100, Covance); rabbit polyclonal antibodies against SrHomer (1:200); and rat Flotillin 1 (1:100, Abcam).

Techniques: Binding Assay, Incubation, SDS Page, In Vitro, Staining, Marker

Journal: International Journal of Molecular Sciences

Article Title: Antimicrobial Compounds in Food Packaging

doi: 10.3390/ijms24032457

Figure Lengend Snippet: Examples of antimicrobial metals application for food preservation.

Article Snippet: Lemongrass oil , ALG edible films , antimicrobial activity of ALG-based films against E. coli ATCC 25922 was not dependent on the type of encapsulating agent; lemongrass oil-ALG film has MIC 0.5%, while ALG film 0.6% , [ ] .

Techniques: Preserving, Polymer, Activity Assay, Bacteria, Modification, Inhibition, Starch

Journal: International Journal of Molecular Sciences

Article Title: Antimicrobial Compounds in Food Packaging

doi: 10.3390/ijms24032457

Figure Lengend Snippet: Examples of antimicrobial metal oxides used in food packaging industry.

Article Snippet: Lemongrass oil , ALG edible films , antimicrobial activity of ALG-based films against E. coli ATCC 25922 was not dependent on the type of encapsulating agent; lemongrass oil-ALG film has MIC 0.5%, while ALG film 0.6% , [ ] .

Techniques: Polymer, In Vitro, Activity Assay, Inhibition, Proximity Ligation Assay, Diffusion-based Assay

Journal: International Journal of Molecular Sciences

Article Title: Antimicrobial Compounds in Food Packaging

doi: 10.3390/ijms24032457

Figure Lengend Snippet: Examples of AMPs and bacteriocins application in active food packaging systems.

Article Snippet: Lemongrass oil , ALG edible films , antimicrobial activity of ALG-based films against E. coli ATCC 25922 was not dependent on the type of encapsulating agent; lemongrass oil-ALG film has MIC 0.5%, while ALG film 0.6% , [ ] .

Techniques: Polymer, Starch, In Vitro, Activity Assay, Bacteria, Modification, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Antimicrobial Compounds in Food Packaging

doi: 10.3390/ijms24032457

Figure Lengend Snippet: Examples of antimicrobial agents of plant origin and their application for food preservation.

Article Snippet: Lemongrass oil , ALG edible films , antimicrobial activity of ALG-based films against E. coli ATCC 25922 was not dependent on the type of encapsulating agent; lemongrass oil-ALG film has MIC 0.5%, while ALG film 0.6% , [ ] .

Techniques: Preserving, Polymer, Bacteria, Activity Assay, Comparison, Starch, Concentration Assay, Inhibition, Diffusion-based Assay, Control, Membrane, Plant Extract

Journal: Cell reports. Physical science

Article Title: Structure-function-guided design of synthetic peptides with anti-infective activity derived from wasp venom

doi: 10.1016/j.xcrp.2023.101459

Figure Lengend Snippet: Amino acid sequence, theoretical values of normalized hydrophobicity, normalized hydrophobic moment, net charge, ratio of polar/non-polar amino acid residues, propensity to in vitro aggregation, amphiphilicity index, and values of helical fraction in water and in a mixture 3:2 of TFE and water, for WT1, WT2, and their corresponding Ala-Scan analogs

Article Snippet: As previously discussed, when Gly at position 12 of WT1 was replaced by Ala, the resulting analog, A 12 -1, was more active than WT1 against E. coli ATCC 11775 and S. aureus ATCC 12600 (MIC = 2 μmol L −1 ) but showed no significant improvement in activity against P. aeruginosa strains PAO1 and PA14 compared with WT1 ( and ).

Techniques: Sequencing, In Vitro

Journal: Cell reports. Physical science

Article Title: Structure-function-guided design of synthetic peptides with anti-infective activity derived from wasp venom

doi: 10.1016/j.xcrp.2023.101459

Figure Lengend Snippet: (A) Schematic representation of the structure-function relationship studies, from the selection of the templates (EMP-EM1 [WT1] and EMP-EM2 [WT2]), isolated from the venom of the solitary wasp Eumenes micado , to the design of an optimized second-generation peptide. (B) Antimicrobial activity of WT1 and WT2 and Ala-Scan analogs for the four pathogenic bacterial strains tested in this study. The red color represents bacterial growth inhibition, and the blue color represents bacterial growth. (C and D) CD spectra of WT1 (C) and WT2 (D) and their respective Ala-Scan derivatives at 50 μmol L −1 in TFE:water 3:2 v/v and water showing the conformational transition of the peptides from random coil in water to α helix in TFE:water. CD spectra were recorded in three replicates at 25°C, using a quartz cuvette with 1-mm path length, between 260 and 190 nm at 50 nm min −1 , with a bandwidth of 0.5 nm. (E) Bidimensional helical wheel representations of the wild-type peptides WT1 and WT2, indicating positions where Ala-substitution decreased (blue arrows) or enhanced (red arrows) activity.

Article Snippet: As previously discussed, when Gly at position 12 of WT1 was replaced by Ala, the resulting analog, A 12 -1, was more active than WT1 against E. coli ATCC 11775 and S. aureus ATCC 12600 (MIC = 2 μmol L −1 ) but showed no significant improvement in activity against P. aeruginosa strains PAO1 and PA14 compared with WT1 ( and ).

Techniques: Selection, Isolation, Activity Assay, Inhibition, Circular Dichroism

Journal: Cell reports. Physical science

Article Title: Structure-function-guided design of synthetic peptides with anti-infective activity derived from wasp venom

doi: 10.1016/j.xcrp.2023.101459

Figure Lengend Snippet: Properties of peptides WT1, WT2, and their corresponding second-generation analogs

Article Snippet: As previously discussed, when Gly at position 12 of WT1 was replaced by Ala, the resulting analog, A 12 -1, was more active than WT1 against E. coli ATCC 11775 and S. aureus ATCC 12600 (MIC = 2 μmol L −1 ) but showed no significant improvement in activity against P. aeruginosa strains PAO1 and PA14 compared with WT1 ( and ).

Techniques: Sequencing, In Vitro

Journal: Cell reports. Physical science

Article Title: Structure-function-guided design of synthetic peptides with anti-infective activity derived from wasp venom

doi: 10.1016/j.xcrp.2023.101459

Figure Lengend Snippet: (A) Antimicrobial activity of WT1, WT2, and second-generation analogs for all tested pathogenic bacteria. The red color represents bacterial growth inhibition, and the blue color represents bacterial growth. Heat maps obtained directly from OD measurements of 96-well plates after treatment are shown in . (B) CD spectra of WT1 and WT2 and their respective second-generation derivatives at 50 μmol L −1 in TFE:water 3:2 v/v, showing α helix conformation, and in water, showing random-coil conformation. CD spectra were recorded in three replicates at 25°C, using a quartz cuvette with 1-mm path length, between 260 and 190 nm at 50 nm min −1 , with a bandwidth of 0.5 nm.

Article Snippet: As previously discussed, when Gly at position 12 of WT1 was replaced by Ala, the resulting analog, A 12 -1, was more active than WT1 against E. coli ATCC 11775 and S. aureus ATCC 12600 (MIC = 2 μmol L −1 ) but showed no significant improvement in activity against P. aeruginosa strains PAO1 and PA14 compared with WT1 ( and ).

Techniques: Activity Assay, Bacteria, Inhibition, Circular Dichroism

Journal: Cell reports. Physical science

Article Title: Structure-function-guided design of synthetic peptides with anti-infective activity derived from wasp venom

doi: 10.1016/j.xcrp.2023.101459

Figure Lengend Snippet: (A) Schematic representation of the NPN assay, in which molecules of NPN (represented by gray spheres) present weak fluorescence emission intensity in an aqueous environment. When the outer membranes are permeabilized by peptides, the NPN molecules interact with the lipidic environment of damaged outer membranes and the intensity of blue fluorescence emission increases (represented by blue spheres). (B) NPN graph for outer membrane permeabilization of Pseudomonas aeruginosa PAO1 by polymyxin B (PMB), WT1, K 12 -1, K 13 -1, WT2, and K 13 -2 peptides. Profiles with a rapid increase in fluorescence emission intensity, followed by a slow decay, were obtained after measurement of white 96-well plates on a Thermo Scientific Varioskan LUX fluorescence spectrophotometer, with the excitation wavelength set to 350 nm and the emission wavelength set to 420 nm, according to the experimental procedure described in the section “ .” All NPN assays were done in three replicates, including the controls, which consisted of only HEPES solution, HEPES solution and NPN (not shown), HEPES solution and P. aeruginosa PAO1 (not shown), and HEPES solution with both P. aeruginosa PAO1 and NPN. Data are represented as mean ± SD. (C) Schematic representation of the DiSC 3 -5 assay, in which molecules of DiSC 3 -5 (represented by gray spheres) accumulate in cytoplasmic membranes and aggregate at high concentrations, causing fluorescence quenching. When the cytoplasmic membrane is destabilized by peptides, DiSC 3 -5 migrates to the cytoplasm or to the external environment, and red fluorescence emission intensity (represented by red spheres) increases. (D) DiSC 3 -5 graph for cytoplasmic membrane depolarization of P. aeruginosa PAO1 by PMB, WT1, K 12 -1, K 13 -1, WT2, and K 13 -2 peptides. Profiles with increases and decreases in fluorescence emission intensity were obtained after measurement of black 96-well plates on a Thermo Scientific Varioskan LUX fluorescence spectrophotometer, with the excitation wavelength set to 622 nm and emission wavelength set to 670 nm as described in the section “ .” DiSC 3 -5 graph obtained after the addition of triton solution is shown in . All DiSC 3 -5 assays were done in three replicates, including the controls, which consisted of only HEPES solution, and HEPES solution containing PAO1 and DiSC 3 -5. Data are represented as mean ± SD. (E) Synergy assay for activity against P. aeruginosa PAO1 between ciprofloxacin, ofloxacin, gentamicin, polymyxin B, or erythromycin, and each of four peptides: WT1, WT2, K 12 -1, and K 13 -1. The Fractional Inhibitory Concentration Index (FICI) values, which indicate the degree of synergy between two antimicrobial agents against a target microorganism, were calculated based on the MICs of WT1, WT2, K 12 -1, and K 13 -1 and the commercial antibiotics used alone and in combination. FICI values <0.5 indicate synergy; 0.5 < FICI < 1 indicates additive effects; 1 < FICI < 4 indicates indifference; and FICI > 4 indicates antagonism (not represented in the graph). (F) Resistance assay: development of resistance to ciprofloxacin, PMB, WT1, K 12 -1, and K 13 -1 in Escherichia coli Δ mutS . The experiment was performed for 20 days as described in detail in the section “ .” Data are represented as mean ± SD.

Article Snippet: As previously discussed, when Gly at position 12 of WT1 was replaced by Ala, the resulting analog, A 12 -1, was more active than WT1 against E. coli ATCC 11775 and S. aureus ATCC 12600 (MIC = 2 μmol L −1 ) but showed no significant improvement in activity against P. aeruginosa strains PAO1 and PA14 compared with WT1 ( and ).

Techniques: NPN Assay, Fluorescence, Membrane, Spectrophotometry, Activity Assay, Concentration Assay

Journal: Cell reports. Physical science

Article Title: Structure-function-guided design of synthetic peptides with anti-infective activity derived from wasp venom

doi: 10.1016/j.xcrp.2023.101459

Figure Lengend Snippet: (A and B) Cytotoxic activity of WT1, A 14 -1, K 12 -1, K 13 -1, WT2, K 10 -2, and K 13 -2 against (A) human embryonic kidney cells (HEK293T) and (B) primary human keratinocytes. (C) Schematic representation of the in vivo assay procedure. The mice were anesthetized with isoflurane and weighed; their backs were shaved, and a superficial linear skin abrasion was made using a needle to damage the stratum corneum and upper layer of the epidermis. Then 50 μL of 10 7 CFU mL −1 in phosphate-buffered saline (PBS) of P. aeruginosa PAO1 was inoculated over the scratch in the back of the mice. After 1 h, peptide solutions in PBS at 32 μmol L −1 for K 13 -2 and 16 μmol L −1 for K 12 -1 and K 13 -1 were added to the infected area. This procedure was done for four mice per peptide tested. After 2 days, mice from each group were killed and weighed, and the area of scarified skin was cut, homogenized using a bead beater for 20 min (25 Hz), and serially diluted for CFU quantification. This procedure was repeated after 4 days with the mice from each group. Two technical replicates were performed for each sample to ensure accuracy. (D) Mice weight monitoring for potential in vivo toxicity assessment. The body weight of infected mice was normalized by the body weight of uninfected mice. Data are represented as mean ± SD. (E) Anti-infective activity of K 12 -1, K 13 -1, and K 13 -2 in vivo compared with control groups. Statistical significance was determined using one-way ANOVA followed by Dunnett’s test; p values are shown in the graph.

Article Snippet: As previously discussed, when Gly at position 12 of WT1 was replaced by Ala, the resulting analog, A 12 -1, was more active than WT1 against E. coli ATCC 11775 and S. aureus ATCC 12600 (MIC = 2 μmol L −1 ) but showed no significant improvement in activity against P. aeruginosa strains PAO1 and PA14 compared with WT1 ( and ).

Techniques: Activity Assay, In Vivo, Saline, Infection, Control

Journal: Scientific Reports

Article Title: Intracellular biomass flocculation as a key mechanism of rapid bacterial killing by cationic, amphipathic antimicrobial peptides and peptoids

doi: 10.1038/s41598-017-16180-0

Figure Lengend Snippet: Calcein leakage after 5 minutes from ( A ) anionic, bacteria-mimetic POPE/POPG (7:3) LUVs and ( B ) zwitterionic, erythrocyte-mimetic POPC/cholesterol (2:1) LUVs. ( C ) Depolarization of E. coli ATCC 35218 after 5-min. treatment, as monitored by diSC3-5 fluorescence. The data are representative of 3 independent experiments.

Article Snippet: The top portion of Table lists the two peptides (pexiganan, an analog of magainin, and bee venom-derived melittin, whose membrane-disruptive activities are well-documented , ) and the peptoids used for these studies, along with their sequences (see Fig. for a guide to peptoid monomers), antibacterial activities against E. coli ATCC 35218, and hemolytic activities.

Techniques: Bacteria, Fluorescence

Journal: Scientific Reports

Article Title: Intracellular biomass flocculation as a key mechanism of rapid bacterial killing by cationic, amphipathic antimicrobial peptides and peptoids

doi: 10.1038/s41598-017-16180-0

Figure Lengend Snippet: In vitro activities of peptoids and peptides.

Article Snippet: The top portion of Table lists the two peptides (pexiganan, an analog of magainin, and bee venom-derived melittin, whose membrane-disruptive activities are well-documented , ) and the peptoids used for these studies, along with their sequences (see Fig. for a guide to peptoid monomers), antibacterial activities against E. coli ATCC 35218, and hemolytic activities.

Techniques: In Vitro, Sequencing

Journal: Scientific Reports

Article Title: Intracellular biomass flocculation as a key mechanism of rapid bacterial killing by cationic, amphipathic antimicrobial peptides and peptoids

doi: 10.1038/s41598-017-16180-0

Figure Lengend Snippet: Scanning electron micrographs of E. coli either ( A ) without treatment, or treated for one hour with 10 μM ( B ) peptoid 1 ( C ) 1-Pro 6 , ( D ) 1 17mer , ( E ) peptoid 2, ( F ) 1 17mer alone (no bacteria), ( G ) pexiganan, or ( H ) melittin. Magnification = 50,000X.

Article Snippet: The top portion of Table lists the two peptides (pexiganan, an analog of magainin, and bee venom-derived melittin, whose membrane-disruptive activities are well-documented , ) and the peptoids used for these studies, along with their sequences (see Fig. for a guide to peptoid monomers), antibacterial activities against E. coli ATCC 35218, and hemolytic activities.

Techniques: Bacteria

Journal: Scientific Reports

Article Title: Intracellular biomass flocculation as a key mechanism of rapid bacterial killing by cationic, amphipathic antimicrobial peptides and peptoids

doi: 10.1038/s41598-017-16180-0

Figure Lengend Snippet: Comparison of transmission electron micrographs of transverse thin sections of representative E. coli treated for 1 hour with enough peptide or peptoid to kill some, but not all of the bacteria in the sample. ( A ) No treatment, ( B ) 10 µM pexiganan, ( C ) 100 µM melittin, ( D ) 10 µM fowlicidin-1, ( E ) 10 µM LL-37, ( F ) 10 µM peptoid 1, ( G ) 100 µM 1- N Lys 5,11 , ( H ) 100 µM 1-Pro 6 , ( I ) 10 µM 1 17mer . Images of bacteria treated with 2, 1 achiral , and 1- N sna 6,12 , are shown in Supp. Fig. . Scale bar represents 100 nm.

Article Snippet: The top portion of Table lists the two peptides (pexiganan, an analog of magainin, and bee venom-derived melittin, whose membrane-disruptive activities are well-documented , ) and the peptoids used for these studies, along with their sequences (see Fig. for a guide to peptoid monomers), antibacterial activities against E. coli ATCC 35218, and hemolytic activities.

Techniques: Comparison, Transmission Assay, Bacteria

Journal: Scientific Reports

Article Title: Intracellular biomass flocculation as a key mechanism of rapid bacterial killing by cationic, amphipathic antimicrobial peptides and peptoids

doi: 10.1038/s41598-017-16180-0

Figure Lengend Snippet: Transmission electron micrographs demonstrating the co-incidence of altered and control-like morphologies in partially killed samples treated with ( A ) 10 µM LL-37 and ( B ) 10 µM peptoid 1. Soft x-ray tomography of E. coli . ( C ) Control (untreated) and ( D ) treated with 10 µM peptoid 1. Scale bar (TEM) represents 100 nm.

Article Snippet: The top portion of Table lists the two peptides (pexiganan, an analog of magainin, and bee venom-derived melittin, whose membrane-disruptive activities are well-documented , ) and the peptoids used for these studies, along with their sequences (see Fig. for a guide to peptoid monomers), antibacterial activities against E. coli ATCC 35218, and hemolytic activities.

Techniques: Transmission Assay, Control, Tomography

Journal: Scientific Reports

Article Title: Intracellular biomass flocculation as a key mechanism of rapid bacterial killing by cationic, amphipathic antimicrobial peptides and peptoids

doi: 10.1038/s41598-017-16180-0

Figure Lengend Snippet: Transmission electron micrographs of E. coli treated with 100 µM peptoid 1 for ( A ) 5 minutes, ( B ) 15 minutes, ( C ) 30 minutes, and ( D ) 1 hour. 1 × 10 4 CFU/mL bacteria remained (0.01% of bacteria in the control) after 5-min treatment; all bacteria were killed after 15-min, 30-min, and 60-min treatments. Scale bar represents 100 nm.

Article Snippet: The top portion of Table lists the two peptides (pexiganan, an analog of magainin, and bee venom-derived melittin, whose membrane-disruptive activities are well-documented , ) and the peptoids used for these studies, along with their sequences (see Fig. for a guide to peptoid monomers), antibacterial activities against E. coli ATCC 35218, and hemolytic activities.

Techniques: Transmission Assay, Bacteria, Control

Journal: Animals : an Open Access Journal from MDPI

Article Title: In Vitro Characterization of Indigenous Probiotic Strains Isolated from Colombian Creole Pigs

doi: 10.3390/ani10071204

Figure Lengend Snippet: In vitro screening of antibacterial activity of Lactobacillus spp. isolates from the native Zungo Pelado breed of pigs against enteropathogenic bacteria ( n = 3).

Article Snippet: The CAM6 strain showed in vitro antibacterial activity against selected enteropathogenic bacteria ( Escherichia coli strain NBRC 102203 and Salmonella enterica serovar Typhimurium 4.5.12) and commensal bacteria ( Klebsiella pneumoniae ATCC BAA-1705D-5 and Pseudomonas aeruginosa ATCC 15442) and resistance to all antibiotics except amoxicillin.

Techniques: In Vitro, Activity Assay, Bacteria, Inhibition

Journal: Molecular and Cellular Biology

Article Title: The Activity and Stability of the Transcriptional Coactivator p/CIP/SRC-3 Are Regulated by CARM1-Dependent Methylation

doi: 10.1128/mcb.00815-06

Figure Lengend Snippet: FIG. 1. CARM1 associates with p/CIP on DNA. (A) HeLa nuclear extracts were separated by gel filtration chromatography, and fractions were assayed by immunoblotting using the antibodies indicated on the left. (B) Fractions corresponding to peaks I and II shown in panel A were pooled and passed through an anti-p/CIP immunoaffinity column or immunoglobulin G (IgG) column as control. After extensive washing, bound proteins were eluted using low pH buffer and assayed by immunoblotting using antibodies specific for p/CIP, CBP, or CARM1. (C) p/CIP and CARM1 bind to the endogenous PS2 promoter in vivo. ChIP analysis of the PS2 promoter in MCF-7 cells using antibodies against p/CIP and CARM1 demonstrates that both p/CIP and CARM1 are recruited to the PS2 promoter in response to estradiol. MCF-7 cells were stimulated for various time periods. Cells were fixed, and sequential ChIPs were performed using an anti-p/CIP antibody, followed by reimmunoprecipitation (ReChIP) using a CARM1-specific antibody. Samples were analyzed by PCR using specific primers flanking the PS2 promoter, and the relative abundance was quantified by densitometry.

Article Snippet: Antibodies against CARM1 and CBP were purchased from Santa Cruz Biotechnologies.

Techniques: Chromatography, Western Blot, Control, In Vivo

Journal: Molecular and Cellular Biology

Article Title: The Activity and Stability of the Transcriptional Coactivator p/CIP/SRC-3 Are Regulated by CARM1-Dependent Methylation

doi: 10.1128/mcb.00815-06

Figure Lengend Snippet: FIG. 2. In vitro methylation of p/CIP. (A) Epitope-tagged recombi- nant proteins were generated in Sf9 cells using baculovirus. Cells were then harvested, and proteins were immunopurified using anti-FLAG Sepharose. Approximately 500 ng of various purified proteins was then analyzed by SDS-PAGE and Coomassie blue staining. (B) Approximately 500 ng of purified recombinant CBP, p/CIP, or specific histones was incubated in the presence of 100 ng of either CARM1 or PRMT1 and [3H]SAM for 1 h. Proteins were then separated by SDS-PAGE and analyzed by fluorography. (C) In vitro methylation assays were performed using 500 ng of p/CIP, 100 ng of recombinant CARM1, and increasing concentrations of histones as indicated at the top of the panel. The reactions were terminated, proteins were separated by SDS-PAGE, and methylation of p/CIP was monitored by fluorography. BSA, bovine serum albumin.

Article Snippet: Antibodies against CARM1 and CBP were purchased from Santa Cruz Biotechnologies.

Techniques: In Vitro, Methylation, Generated, SDS Page, Staining, Recombinant, Incubation

Journal: Molecular and Cellular Biology

Article Title: The Activity and Stability of the Transcriptional Coactivator p/CIP/SRC-3 Are Regulated by CARM1-Dependent Methylation

doi: 10.1128/mcb.00815-06

Figure Lengend Snippet: FIG. 3. p/CIP is methylated in intact cells. (A) Extracts from CARM1/ MEFs do not support methylation of p/CIP in vitro. Ap- proximately 500 ng of purified recombinant p/CIP protein was incu- bated with extracts derived from either CARM1/ or CARM1/

Article Snippet: Antibodies against CARM1 and CBP were purchased from Santa Cruz Biotechnologies.

Techniques: Methylation, In Vitro, Recombinant, Derivative Assay

Journal: Molecular and Cellular Biology

Article Title: The Activity and Stability of the Transcriptional Coactivator p/CIP/SRC-3 Are Regulated by CARM1-Dependent Methylation

doi: 10.1128/mcb.00815-06

Figure Lengend Snippet: FIG. 4. Molecular mapping of CARM1-dependent methylation of p/CIP. p/CIP truncation mutants were generated using a baculovirus system and purified by immunoaffinity chromatography. Approximately 500 ng of the various truncation mutants was then tested as a substrate for CARM1 by in vitro methylation assay using 100 ng of purified CARM1. On the left is the Coomassie-stained gel of the purified proteins resolved on an 8% SDS-PAGE gel, and on the right is the corresponding fluorograph. (B) One microgram of biotinylated peptides corresponding to the regions of p/CIP indicated was used as a substrate, and the incorporation of [3H]SAM was measured by in vitro methylation in the presence of purified CARM1. The asterisk indicates the three dimethylated arginines identified by mass spectrometry. (C) Homology of MD1 within the SRC family of proteins. SRC3 corresponds to the human homologue of p/CIP. The shaded areas indicate conserved arginines, and asterisks indicate arginines identified by mass spectrometry in p/CIP.

Article Snippet: Antibodies against CARM1 and CBP were purchased from Santa Cruz Biotechnologies.

Techniques: Methylation, Generated, Chromatography, In Vitro, Staining, SDS Page, Mass Spectrometry

Journal: Molecular and Cellular Biology

Article Title: The Activity and Stability of the Transcriptional Coactivator p/CIP/SRC-3 Are Regulated by CARM1-Dependent Methylation

doi: 10.1128/mcb.00815-06

Figure Lengend Snippet: FIG. 5. Phosphorylation at S847 antagonizes methylation of p/CIP at R839. (A) GST recombinant proteins containing various regions of p/CIP spanning the full-length protein were generated in bacteria, purified, and used as substrates (500 ng) for purified CARM1 (100 ng) by in vitro methylation. At left is a Coomassie-stained gel of the GST fusion proteins used in the methylation assay resolved on a 12% SDS-PAGE gel, and at right is the corresponding fluorograph of the in vitro methylation reactions. (B) Comparison of the MD2 domains between p/CIP and SRC3. The asterisk corresponds to the phosphorylation site at aa 847. The shaded areas contain the methylation sites defined by in vitro methylation reactions using recombinant GST proteins. (C) Biotinylated peptides corresponding to the regions P1, P2, and P3 (shaded areas in panel B) were used as substrates, and the incorporation of [3H]SAM was measured by in vitro methylation in the presence of purified CARM1. Peptides corresponding to P1R839A and P1R844A contain substitutions of arginine to alanine. The peptide P1S847(p) is identical to P1 but contains a phosphorylated serine at aa 847.

Article Snippet: Antibodies against CARM1 and CBP were purchased from Santa Cruz Biotechnologies.

Techniques: Phospho-proteomics, Methylation, Recombinant, Generated, Bacteria, In Vitro, Staining, SDS Page, Comparison

Journal: Molecular and Cellular Biology

Article Title: The Activity and Stability of the Transcriptional Coactivator p/CIP/SRC-3 Are Regulated by CARM1-Dependent Methylation

doi: 10.1128/mcb.00815-06

Figure Lengend Snippet: FIG. 6. The relative abundance of p/CIP is regulated by arginine methylation. Epitope-tagged recombinant wild-type p/CIP (wt), or p/CIP mutants containing arginine substitutions at CARM1 methylation sites (p/CIPR3A or p/CIPR6A) were generated in Sf9 cells using baculovirus. Cells were then harvested, and proteins were immunopurified using anti-FLAG-Sepharose. Approximately 1 g of each purified protein was analyzed by SDS-PAGE and Coomassie blue staining (left) and used as substrates for in vitro CARM1-dependent methylation reactions (right). (B) COS-1 cells were transfected with expression vectors for wild-type p/CIP (wt), p/CIPR3A, or p/CIPR6A. After 24 h cells were lysed, and whole-cell extracts were prepared. Equal amounts of protein from each extract were then analyzed by Western blotting using an anti-FLAG antibody for p/CIP or anti-tubulin antibody (left). Transfected samples were immunopurified using anti-FLAG-Sepharose, followed by immuno- blotting with anti-FLAG antibody (right). (C) CARM1/ or CARM1/ MEFs were incubated with [35S]methionine for 40 min, followed by incubation with nonradioactive methionine-containing medium for the indicated time periods. Cells were then lysed, and p/CIP was immunopre- cipitated and then subjected to SDS-PAGE and fluorography. (D) U2OS cells were transfected with either p/CIP wild-type or p/CIP R6A mutant. After 24 h, cells were incubated with [35S]methionine for 40 min and then incubated with cold methionine-containing medium for the indicated time periods. Cells were then lysed, and p/CIP was immunoprecipitated using anti-FLAG-Sepharose and then subjected to SDS-PAGE and fluorography. Samples were quantified by densitometry and expressed as a percentage of p/CIP remaining normalized to time zero.

Article Snippet: Antibodies against CARM1 and CBP were purchased from Santa Cruz Biotechnologies.

Techniques: Methylation, Recombinant, Generated, SDS Page, Staining, In Vitro, Transfection, Expressing, Western Blot, Incubation, Mutagenesis, Immunoprecipitation

Journal: Molecular and Cellular Biology

Article Title: The Activity and Stability of the Transcriptional Coactivator p/CIP/SRC-3 Are Regulated by CARM1-Dependent Methylation

doi: 10.1128/mcb.00815-06

Figure Lengend Snippet: FIG. 7. Methylation of p/CIP is associated with increased degradation. HeLa cells were grown in the presence of AdOx for 8 days, followed by immunoprecipitation of p/CIP. Equal concentrations of p/CIP were then incubated with extracts from CARM1/ or CARM1/ MEFs in the presence of SAM for the indicated time periods. Reactions were then terminated and analyzed by Western blotting using anti-p/CIP antibody. The graph on the right indicates the relative amounts of p/CIP remaining, based on densitometry.

Article Snippet: Antibodies against CARM1 and CBP were purchased from Santa Cruz Biotechnologies.

Techniques: Methylation, Immunoprecipitation, Incubation, Western Blot

Journal: Molecular and Cellular Biology

Article Title: The Activity and Stability of the Transcriptional Coactivator p/CIP/SRC-3 Are Regulated by CARM1-Dependent Methylation

doi: 10.1128/mcb.00815-06

Figure Lengend Snippet: FIG. 9. The association between CBP and p/CIP is decreased in CARM1/ MEFs. (A) Whole-cell extracts were prepared from CARM1/ MEFs or CARM1/ MEFs (at a similar passage num- ber). Approximately 20 g of protein from each extract was separated by SDS-PAGE, and Western blotting was performed using anti-p/CIP antibody or antitubulin as a control. (B) Whole-cell extracts were prepared from CARM1/ MEFs or CARM1/ MEFs, and the amount of starting material in the input was normalized so that each had approximately the same amount of p/CIP based on Western blot- ting. p/CIP was then immunopurified by passing the protein extracts through an anti-p/CIP immunoaffinity column. After extensive wash- ing, the bound proteins were eluted using 100 mM glycine (pH 3.0). Fractions were collected and assayed for p/CIP or CBP by SDS-PAGE followed by Western blotting.

Article Snippet: Antibodies against CARM1 and CBP were purchased from Santa Cruz Biotechnologies.

Techniques: SDS Page, Western Blot, Control

Journal: Frontiers in Microbiology

Article Title: Lily polysaccharides alleviate colitis through the microbiota–N8-acetylspermidine–cGAS–STING signaling axis

doi: 10.3389/fmicb.2025.1686902

Figure Lengend Snippet: The therapeutic effect of lily polysaccharide on colitis. (a) Changes of mice weight. (Day x/Day 0 *100%, n = 5). (b) Disease activity index. ( n = 4). (c,d) Quantified lengths of colonic tissues isolated from mice after treatment ( n = 5). (e) H&E-stained histological sections of colons. Representative digital photos. (f) The pathology score of colons from mice in the different treatment group ( n = 5). (g–i) The levels of pro-inflammatory cytokines IL-1 β , IL-6 and TNF- α in colon of mice were detected by ELISA ( n = 5). (j) The expression levels of MUC2 were determined by immunofluorescence staining in the mouse colon. Representative digital photos. Data are presented as the mean ± SEM. Statistically significant differences are indicated; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Subsequently, the samples were washed again with PBST and incubated overnight at 4 °C with a primary antibody against MUC2 (Rabbit polyclonal IgG, Proteintech, Catalog No. 27675-1-AP, unconjugated; 1:200 dilution).

Techniques: Activity Assay, Isolation, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence

Journal: Frontiers in Microbiology

Article Title: Lily polysaccharides alleviate colitis through the microbiota–N8-acetylspermidine–cGAS–STING signaling axis

doi: 10.3389/fmicb.2025.1686902

Figure Lengend Snippet: Fecal bacteria transplantation experiments showed that LP could positively regulate the gut microbiota. (a) Changes of mice weight. (Day x/Day 0 *100%, n = 5). (b) Disease activity index ( n = 5). (c,d) Quantified lengths of colonic tissues isolated from mice after treatment ( n = 5). (e) H&E-stained histological sections of colons. Representative digital photos. (f) The pathology score of colons from mice in the different treatment group ( n = 5). (g–i) The levels of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in colon of mice were detected by ELISA ( n = 5). (j) The expression levels of MUC2 were determined by immunofluorescence staining in the mouse colon. Representative digital photos. Data are presented as the mean ± SEM. Statistically significant differences are indicated; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Subsequently, the samples were washed again with PBST and incubated overnight at 4 °C with a primary antibody against MUC2 (Rabbit polyclonal IgG, Proteintech, Catalog No. 27675-1-AP, unconjugated; 1:200 dilution).

Techniques: Bacteria, Transplantation Assay, Activity Assay, Isolation, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence

Journal: Frontiers in Microbiology

Article Title: Lily polysaccharides alleviate colitis through the microbiota–N8-acetylspermidine–cGAS–STING signaling axis

doi: 10.3389/fmicb.2025.1686902

Figure Lengend Snippet: Replenishing N8AS can effectively alleviate enteritis. (a) Changes of mice weight.(Day x/Day 0 *100%, n = 5). (b) Disease activity index. ( n = 5). (c,d) Quantified lengths of colonic tissues isolated from mice after treatment ( n = 5). (e) H&E-stained histological sections of colons. Representative digital photos. (f) The pathology score of colons from mice in the different treatment group ( n = 5). (g–i) The levels of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in colon of mice were detected by ELISA ( n = 5). (j) The expression levels of MUC2 were determined by immunofluorescence staining in the mouse colon. Representative digital photos. (k–o) The protein expression levels of cGAS, STING, p-TBK, p-IRF3 were assessed by western blot analysis. Data are presented as the mean ± SEM. Statistically significant differences are indicated; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Subsequently, the samples were washed again with PBST and incubated overnight at 4 °C with a primary antibody against MUC2 (Rabbit polyclonal IgG, Proteintech, Catalog No. 27675-1-AP, unconjugated; 1:200 dilution).

Techniques: Activity Assay, Isolation, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence, Western Blot

Journal: Frontiers in Microbiology

Article Title: Lily polysaccharides alleviate colitis through the microbiota–N8-acetylspermidine–cGAS–STING signaling axis

doi: 10.3389/fmicb.2025.1686902

Figure Lengend Snippet: In vitro verification, lily polysaccharide can inhibit the cGAS-STING pathway and play an anti-inflammatory role. (a) IL-1β mRNA expression. (b) TNF-α mRNA expression level. (c) IL-6 mRNA expression level. (d) ZO-1 mRNA expression level. (e) occludin mRNA expression level. (f) MUC2 mRNA expression level. (g–k) The protein expression levels of cGAS, STING, p-TBK, p-IRF3 were assessed by western blot analysis. Data are presented as the mean ± SEM. Statistically significant differences are indicated; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Subsequently, the samples were washed again with PBST and incubated overnight at 4 °C with a primary antibody against MUC2 (Rabbit polyclonal IgG, Proteintech, Catalog No. 27675-1-AP, unconjugated; 1:200 dilution).

Techniques: In Vitro, Expressing, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Regulation of Two JunD Isoforms by Jun N-terminal Kinases

doi: 10.1074/jbc.m204552200

Figure Lengend Snippet: FIG. 1. Structural organization of JunD and Jun fusion proteins. A, two isoforms of JunD are generated from a single, intron-less mRNA through use of an alternative translational start site. Both isoforms are identical in sequence except for the additional 48 amino acids at the N-terminal transactivation domain of the full-length isoform (JunD-FL) com- pared with the truncated isoform (JunD). Both isoforms contain a region similar in sequence to the JNK docking domain in c-Jun. Three phospho-acceptor residues at positions 90, 100, and 117 are indicated. The basic DNA-binding domain and the leucine-zipper dimerization do- main are labeled. B, the pGEX4T-3 plas- mid was used to generate GST fusions between N-terminal fragments of both JunD-FL and JunD as well as c-Jun. Fusion proteins were expressed and puri- fied from bacteria and used for in vitro kinase and interaction assays. C, GAL4 fusion proteins containing the N-terminal transactivation domains of JunD-FL and JunD were expressed in transfected CHO cells for transcriptional assays.

Article Snippet: Nonspecific interactions were blocked by preincubation of the membranes with 5% nonfat powdered milk in phosphate-buffered saline supplemented with 0.1% Tween 20 for 1 h. GAL4-Jun fusion proteins were detected using -GAL4 mouse monoclonal antibody directed against the GAL4-DNAbinding domain (SC-510; Santa Cruz Biotechnology).

Techniques: Generated, Sequencing, Binding Assay, Labeling, Bacteria, In Vitro, Transfection

Journal: Journal of Biological Chemistry

Article Title: Regulation of Two JunD Isoforms by Jun N-terminal Kinases

doi: 10.1074/jbc.m204552200

Figure Lengend Snippet: FIG. 7. JNK docking domain and target site mutants exhibit lower responsiveness to JNK activation in cultured cells. A, JunD-FL (1–149) and JunD (49–149) were fused to the DNA-binding domain of the yeast transcription factor GAL4 (1–147) using a modified pSG424 expression vector (pGAL0). JNK docking domain mutations (L57A/L59A) and JNK target site mutations (S90A/S100A) were intro- duced into these constructs. These expression plasmids (2 ng each) were co-transfected into CHO cells along with the G5E1bLuc reporter plas- mid (300 ng) in 24-well plates, and the luciferase activity was measured 24 h after transfection. As indicated, expression plasmids for HA- tagged JNKK2 and JNK1 (25 and 13 ng, respectively) were co-trans- fected to augment JNK activity in the cells. These transfections were repeated at least four times. The luciferase activity shown is the aver- age of three replicates in a representative experiment. The bars indicate the standard deviation for each point. B, threonine 117 confers JNK responsiveness to JunD. GAL4-JunD (1–149) containing a single ala- nine substitution at position 117 or the triple mutant S90A/S100A/ S117A were tested as described for A. C, Western blot analysis of cells transfected for transcriptional assays. The indicated GAL4 wild-type (WT) and mutant proteins were detected in whole cell extracts using an anti-GAL4 antibody. Expression of HA-tagged JNK1 and JNKK2 was detected with an anti-HA antibody (lower right panel).

Article Snippet: Nonspecific interactions were blocked by preincubation of the membranes with 5% nonfat powdered milk in phosphate-buffered saline supplemented with 0.1% Tween 20 for 1 h. GAL4-Jun fusion proteins were detected using -GAL4 mouse monoclonal antibody directed against the GAL4-DNAbinding domain (SC-510; Santa Cruz Biotechnology).

Techniques: Activation Assay, Cell Culture, Binding Assay, Modification, Expressing, Plasmid Preparation, Construct, Transfection, Luciferase, Activity Assay, Standard Deviation, Mutagenesis, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Regulation of Two JunD Isoforms by Jun N-terminal Kinases

doi: 10.1074/jbc.m204552200

Figure Lengend Snippet: FIG. 8. Phosphorylation of GAL4-JunD (1–149) by JNK in cul- tured fibroblasts. CHO cells were transfected with GAL-JunD (1– 149), JNK1, and JNKK2 expression plasmids for 24 h, followed by treatment with IL-1 20 min before harvesting to stimulate JNK ac- tivity. GAL4-JunD was immunoprecipitated using a specific anti-GAL4 antibody followed by Western blot analysis. Two antibodies prepared against phospho-c-Jun peptides that cross-react with the corresponding region of phospho-JunD were used: Upstate anti-phospho-c-Jun rabbit polyclonal antibody (antibody 659) (A) and anti-phospho-c-Jun mono- clonal antibody P-2 prepared by Lallemand et al. (43) (B).

Article Snippet: Nonspecific interactions were blocked by preincubation of the membranes with 5% nonfat powdered milk in phosphate-buffered saline supplemented with 0.1% Tween 20 for 1 h. GAL4-Jun fusion proteins were detected using -GAL4 mouse monoclonal antibody directed against the GAL4-DNAbinding domain (SC-510; Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Transfection, Expressing, Immunoprecipitation, Western Blot

Journal: Nature communications

Article Title: MED26 regulates the transcription of snRNA genes through the recruitment of little elongation complex.

doi: 10.1038/ncomms6941

Figure Lengend Snippet: Figure 2 | Reconstitution of LEC by a baculovirus expression system. (a) ICE2 (NARG2) binds to ELL in the presence of ICE1 (KIAA0947). FLAG-immunopurified complexes from baculovirus-infected Sf9 cells expressing the indicated proteins were analysed by western blot. (b) ICE1 binds to EAF1 in the presence of ELL. HA-immunopurified complexes from baculovirus-infected Sf9 cells expressing the indicated proteins were analysed by western blot. (c) ICE2 binds to ELL/EAF1 in the presence of ICE1. FLAG-immunopurified complexes from baculovirus-infected Sf9 cells expressing the indicated proteins were analysed by western blot. (d) Proposed architecture of the components of LEC and model for MED26-NTD function as a docking site for LEC. MED26-NTD interacts with LEC through direct interaction with EAF. In turn, the MED26 CTD binds to Pol II through Mediator. The binding site for MED26-NTD on EAF is represented by black bars. (e) Silver staining of HA-ICE1-CL/FLAG-ICE2/FLAG-ELL/Myc-EAF1 complex reconstituted by the baculovirus expression system. FLAG-tagged ELL, Myc-tagged EAF1, FLAG-tagged ICE2 and HA-tagged ICE1 C-terminal fragments (CL: 1,191–2,266) were co-expressed in Sf9 insect cells, and the ICE1-CL/ICE2/ELL/EAF1 complex was purified by anti-HA affinity chromatography as described in the Methods section. * indicates heavy and light chains derived from anti-HA antibodies used in affinity purification. (f) ICE1-CL/ICE2/ELL/EAF1 complex enhances transcription elongation by Pol II. Oligo(dC)-tailed template transcription assays were performed as described in the Results and Methods sections. Arrowhead indicates the position of the nascent transcript that is synthesized in the presence of ATP, GTP and CTP and stalled at the T site.

Article Snippet: Specific antibodies used were as follows: MED26 (H-228, sc-48776 X, Santa Cruz Biotechnology), MED23 (A300-425A-1, Bethyl Laboratories), ICE1 (raised against bacterially expressed, His-tagged recombinant proteins of ICE1 1,865–2,266 aa), ELL (A301-645A, Bethyl Laboratories), EAF2 (A302-502A-1, Bethyl Laboratories), TAF7 (SQ-8, sc-101167, Santa Cruz Biotechnology), Rpb1 (N20, sc-899, Santa Cruz Biotechnology) and RNA Pol II CTD phospho Ser7 (Cat# 61087, Active motif, Carlsbad, CA).

Techniques: Expressing, Infection, Western Blot, Binding Assay, Silver Staining, Chromatography, Derivative Assay, Synthesized

Journal: Nature communications

Article Title: MED26 regulates the transcription of snRNA genes through the recruitment of little elongation complex.

doi: 10.1038/ncomms6941

Figure Lengend Snippet: Figure 9 | Depletion of TAF7 in cells increases the occupancy of LEC at Pol II-transcribed snRNA genes. (a) Western blot showing TAF7 and Hsp90 at 48 h after transient transfection of non-targeting (control) siRNA pool or siRNA pool targeting TAF7. (b) Effect of TAF7 depletion on transcript levels of Pol II-transcribed snRNA genes detected by RT–qPCR performed with total RNA from cells transfected with siRNAs. Ct values were normalized to GAPDH. Data points are the average of three independent experiments, and error bars show s.d. The P values for the indicated comparisons were determined by Student’s t-test (*Po0.05; **Po0.01). (c) Schematic representation of immobilized oligo(dC)-tailed template assay. Templates immobilized on streptavidin beads were incubated with purified recombinant protein EAF1 in the presence or absence of Mediator and/or TAF7 N243. After washing, bead-bound proteins were detected by western blot. (d) TAF7 interferes with EAF1 recruitment by Mediator in vitro. Upper panel shows immobilized oligo(dC)-tailed template assays. Assays were performed with an oligo(dC)-tailed template, EAF1, and Mediator in the presence or absence of TAF7 N243. Lower panel shows silver staining of Mediator and HA-tagged TAF7 N243 used in the assay. Mediator ( 1) and HA-TAF7 N243 ( 1) contain 1 pmol of the each protein or protein complex. (e) Model of immobilized oligo(dC)-tailed template assay. TAF7 N243 inhibits EAF1 recruitment to the oligo(dC)-tailed template by MED26-containing Mediator. (f) Depletion of TAF7 increased the occupancy of ICE1 (KIAA0947), EAF2 and total Pol II at U4-1 and U5B snRNA genes. Ct values of each ChIP were normalized to that of input. Data points are the average of three independent experiments, and error bars show s.d. The P values for the indicated comparisons were determined by Student’s t-test (*Po0.05; **Po0.01).

Article Snippet: Specific antibodies used were as follows: MED26 (H-228, sc-48776 X, Santa Cruz Biotechnology), MED23 (A300-425A-1, Bethyl Laboratories), ICE1 (raised against bacterially expressed, His-tagged recombinant proteins of ICE1 1,865–2,266 aa), ELL (A301-645A, Bethyl Laboratories), EAF2 (A302-502A-1, Bethyl Laboratories), TAF7 (SQ-8, sc-101167, Santa Cruz Biotechnology), Rpb1 (N20, sc-899, Santa Cruz Biotechnology) and RNA Pol II CTD phospho Ser7 (Cat# 61087, Active motif, Carlsbad, CA).

Techniques: Western Blot, Transfection, Control, Quantitative RT-PCR, Incubation, Recombinant, In Vitro, Silver Staining

Journal: Materials

Article Title: Cationic Substitutions in Hydroxyapatite: Current Status of the Derived Biofunctional Effects and Their In Vitro Interrogation Methods

doi: 10.3390/ma11112081

Figure Lengend Snippet: Synopsis of the bio-functionality realm of cation-substituted hydroxyapatites.

Article Snippet: Ce (3+) , Powder Coating , 4–20 , ○ Induces the in vitro formation of bone-like apatite in SBF; ○ In vitro cytocompatibility with L929 (for Ce-HA dose <100 μg mL −1 ) and MC3T3-E1 cell lines; ○ Cytotoxicity on pulmonary adenocarcinoma (A549) cells in Ce0.1HA, but improvement of cell viability in conjunction with strontium [ ]; ○ Antibacterial effect against S. aureus (ATCC 6538), Lactobacillus (ATCC 393), E. coli (8099), and P. aeruginosa ; enhanced zone inhibition is achieved for Gram-negative E. coli with respect to Gram-positive S. aureus . , [ , , , , , ] .

Techniques: In Vitro, Isolation, In Vivo, Animal Model, Adsorption, Concentration Assay, Modification, Dissolution, Activity Assay, Staining, Bacteria, Control, Transformation Assay