Journal: Journal of Food and Drug Analysis

Article Title: Cannabis sativa L .: A comprehensive review on legislation, decriminalization, phytochemistry, antimicrobial activity, and safety

doi: 10.38212/2224-6614.3471

Figure Lengend Snippet: Antibacterial activity of Cannabis sativa volatile and organic extracts.

Article Snippet: Last but not least, the “Futura” cultivars showed high to moderate activity against the various tested bacteria, with MIC values ranging from 0.625 μL/mL to >20 μL/mL against Enterococcus faecium ATCC 19434, which was consistent with earlier investigations [ ].

Techniques: Activity Assay, Cannabis, Diffusion-based Assay, Inhibition, Bacteria, Antibiofilm Assay, Serial Time-encoded Amplified Microscopy, Distillation, Drug Susceptibility Assay

Journal: Journal of Food and Drug Analysis

Article Title: Cannabis sativa L .: A comprehensive review on legislation, decriminalization, phytochemistry, antimicrobial activity, and safety

doi: 10.38212/2224-6614.3471

Figure Lengend Snippet: Antibacterial activity of Cannabis sativa bioactive compounds.

Article Snippet: Last but not least, the “Futura” cultivars showed high to moderate activity against the various tested bacteria, with MIC values ranging from 0.625 μL/mL to >20 μL/mL against Enterococcus faecium ATCC 19434, which was consistent with earlier investigations [ ].

Techniques: Activity Assay, Cannabis, Staining, Membrane, Diffusion-based Assay, Drug Susceptibility Assay, Motility Assay, Tube Formation Assay, Infection, Inhibition, Bacteria

Journal: MedChemComm

Article Title: Triazole–diindolylmethane conjugates as new antitubercular agents: synthesis, bioevaluation, and molecular docking † †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8md00055g

doi: 10.1039/c8md00055g

Figure Lengend Snippet: In vitro antitubercular activity of compounds 6a–s against the Mtb H37Ra strain

Article Snippet: In a standard primary screening, the newly synthesized triazole–diindolylmethane conjugates 6a–s were tested for their in vitro antitubercular activity against M. tuberculosis H37Ra (ATCC 25177) at three different concentrations, i.e. , 30, 10, and 3 μg mL –1 , using an established XTT reduction menadione assay (XRMA) antitubercular screening protocol, 16 and the results are given in Table S1 (ESI † ).

Techniques: In Vitro, Activity Assay

Journal: Molecular Biology of the Cell

Article Title: The ubiquitin ligase deltex-3l regulates endosomal sorting of the G protein–coupled receptor CXCR4

doi: 10.1091/mbc.e13-10-0612

Figure Lengend Snippet: FIGURE 1: A role for DTX3L in CXCR4 degradation. (A) HeLa cells transfected with siRNA directed against control, AIP4, or DTX3L were treated with vehicle (PBS + 0.1% BSA) or 10 nM CXCL12 for 3 h. Whole-cell lysates were analyzed for the levels of endogenous CXCR4 and the indicated proteins by immunoblotting (IB). (B) CXCR4 levels normalized to actin were determined by densitometric analysis. Data represent the average percentage CXCR4 degraded in CXCL12-treated vs. vehicle-treated cells. Representative immunoblots are shown in A, and B represents quantification of eight independent experiments. Error bars represent SEM. Data were analyzed by a one-way ANOVA (p < 0.0001), followed by Bonferroni’s posthoc test. CXCR4 degradation in AIP4 (p < 0.001) and DTX3L (p < 0.001) siRNA–treated cells was significantly different from siRNA control (siCtrl).

Article Snippet: The mouse monoclonal antibodies directed against HRS (c-19) and ubiquitin (P4D1) and the goat polyclonal directed against DTX3L (N-16) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Control, Western Blot

Journal: Molecular Biology of the Cell

Article Title: The ubiquitin ligase deltex-3l regulates endosomal sorting of the G protein–coupled receptor CXCR4

doi: 10.1091/mbc.e13-10-0612

Figure Lengend Snippet: FIGURE 2: DTX3L localizes to early endosomes upon CXCR4 activation. (A) HeLa cells were treated with vehicle or CXCL12 for 30 min. Cells were then fixed, permeabilized, and incubated with antibodies directed against DTX3L and the early endosomal marker EEA1 or the lysosomal marker LAMP2. Inset represents 3× enlargement of the boxed region. Yellow in the merged image indicates colocalization between DTX3L and EEA1 or LAMP2. Differential interference contrast (DIC) images are shown. Equal acquisition settings (gain and intensity) were used between parallel samples within each experiment. Representative images from four independent experiments. (B) CXCR4 activation increases DTX3L puncta number in HeLa cells. DTX3L puncta were counted using ImageJ software. Data represent the average puncta count from 35–45 cells from four independent experiments. Error bars represent SEM (not visible). (C, D) Pearson product–moment correlation coefficient calculated to determine the level of colocalization between DTX3L and EEA1 (C) and LAMP2 (D). Data were analyzed by Student’s t test.

Article Snippet: The mouse monoclonal antibodies directed against HRS (c-19) and ubiquitin (P4D1) and the goat polyclonal directed against DTX3L (N-16) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Incubation, Marker, Software

Journal: Molecular Biology of the Cell

Article Title: The ubiquitin ligase deltex-3l regulates endosomal sorting of the G protein–coupled receptor CXCR4

doi: 10.1091/mbc.e13-10-0612

Figure Lengend Snippet: FIGURE 3: DTX3L regulates CXCR4 sorting from early endosomes to lysosomes. (A) HeLa cells transfected with siRNA directed against control or DTX3L were treated with vehicle (PBS + 0.1% BSA) or 10 nM CXCL12 for 3 h. Cells were then fixed, permeabilized, and incubated with antibodies directed against CXCR4, DTX3L, early endosomal marker EEA1, or late endosomal/lysosomal marker LAMP2. Inset represents 3× enlargement of the boxed region. Yellow in the merged image indicates colocalization between CXCR4 and EEA1 or LAMP2. DIC images are shown. Equal acquisition settings (gain and intensity) were used between parallel samples within each experiment. Representative images are from four independent experiments. (B) Pearson product–moment correlation coefficient determined using the ImageJ plug-in Colocalization Finder. Data represent the average from four independent experiments. Data were analyzed by a one-way ANOVA (p < 0.001), followed by Bonferroni’s posthoc test. (C) CXCR4 puncta counted using the ImageJ function Analyze Particles. Data represent the average puncta count from 45–60 cells from three independent experiments. Error bars represent SEM. Data were analyzed by Student’s t test.

Article Snippet: The mouse monoclonal antibodies directed against HRS (c-19) and ubiquitin (P4D1) and the goat polyclonal directed against DTX3L (N-16) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Control, Incubation, Marker

Journal: Molecular Biology of the Cell

Article Title: The ubiquitin ligase deltex-3l regulates endosomal sorting of the G protein–coupled receptor CXCR4

doi: 10.1091/mbc.e13-10-0612

Figure Lengend Snippet: FIGURE 4: AIP4 and DTX3L interact directly and form a complex in HeLa cells. (A) Cleared lysates from HeLa cells (500 μg) were incubated with either anti-IgG control or anti-DTX3L antibodies to immunoprecipitate endogenous DTX3L. (B) Equimolar amounts of His-tagged DTX3L (1–10 nM) were incubated with equal amounts of purified GST-AIP4 (100 nmol). Samples were resolved by 10% SDS–PAGE and analyzed by immunoblotting for DTX3L or GST. Input represents 10% of HIS-DTX3L used in the binding reaction. Data represent the fold change in GST-AIP4 binding to HIS-DTX3L. Data are representative of three independent experiments. Data were analyzed by one-way ANOVA, followed by Bonferroni’s posthoc test. Data show a significant increase in His-DTX3L binding to GST-AIP4 with increasing His-DTX3L concentration. (C) HeLa lysates (500 μg) were treated with CXCL12 (10 nM) and incubated with either anti-IgG control or anti-DTX3L antibodies to immunoprecipitate endogenous DTX3L. Samples were resolved by 10% SDS–PAGE and analyzed by immunoblotting with the indicated antibodies. The amount of AIP4 binding was quantitated by densitometric analysis from four independent experiments and then analyzed by one-way ANOVA, followed by followed by Bonferroni’s posthoc test for significance (p < 0.05). Error bars represent the SEM. (D) HeLa cells transfected with FLAG-tagged AIP4 were treated with 10 nM CXCL12 for 0–60 min. Cells were fixed, permeabilized, and incubated with antibodies directed against FLAG-AIP4, DTX3L, and EEA1. Representative images are from three independent experiments. DIC images are shown. Inset, 3× enlargement of the boxed region. Equal acquisition settings (gain and intensity) were used between parallel samples within each experiment. (E) Immunoblot showing level of FLAG-AIP4 expression over endogenous AIP4. (F) FLAG-AIP4 and DTX3L show strong colocalization, as determined by calculating the Pearson product–moment correlation coefficient. (G, H) Puncta were counted using the particle analysis software of ImageJ. Data represent the average FLAG-AIP4 (G) or DTX3L (H) puncta per cell from four independent experiments. Data were analyzed by one-way ANOVA, followed by Bonferroni’s posthoc test.

Article Snippet: The mouse monoclonal antibodies directed against HRS (c-19) and ubiquitin (P4D1) and the goat polyclonal directed against DTX3L (N-16) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Incubation, Control, Purification, SDS Page, Western Blot, Binding Assay, Concentration Assay, Transfection, Expressing, Particle Size Analysis, Software

Journal: Molecular Biology of the Cell

Article Title: The ubiquitin ligase deltex-3l regulates endosomal sorting of the G protein–coupled receptor CXCR4

doi: 10.1091/mbc.e13-10-0612

Figure Lengend Snippet: FIGURE 6: DTX3L regulates the distribution of HRS and STAM1. (A, D) HeLa cells transfected with either control or DTX3L siRNA were treated with 10 nM CXCL12 for 30 min. Cells were fixed, permeabilized, and incubated with antibodies directed against DTX3L, HRS, (A) and STAM-1 (D). Inset, 3× enlargement of the boxed region. Yellow in the merged image indicates colocalization between HRS (A) or STAM-1 (D) and DTX3L. DIC images are shown. Equal acquisition settings (gain and intensity) were used between parallel samples within each experiment. (B, E) HRS and STAM-1 puncta are reduced in DTX3L siRNA–treated cells. Puncta were counted using the particle analysis software of ImageJ. Data represent the average HRS (B) or STAM-1 (E) puncta per cell from four and three independent experiments, respectively. For the HRS puncta analysis, 110–145 cells were examined, and for the STAM-1 puncta analysis, 45–50 cells were examined. Data were analyzed by Student’s t test. DTX3L shows strong colocalization with HRS (C) and STAM-1 (F) in control siRNA–treated cells, as determined by calculating the Pearson product–moment correlation coefficient.

Article Snippet: The mouse monoclonal antibodies directed against HRS (c-19) and ubiquitin (P4D1) and the goat polyclonal directed against DTX3L (N-16) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Transfection, Control, Incubation, Particle Size Analysis, Software

Journal: Molecular Biology of the Cell

Article Title: The ubiquitin ligase deltex-3l regulates endosomal sorting of the G protein–coupled receptor CXCR4

doi: 10.1091/mbc.e13-10-0612

Figure Lengend Snippet: FIGURE 7: DTX3L inhibits the E3 ubiquitin ligase activity of AIP4. (A) Purified AIP4, AIP4-C830A, HIS-DTX3L, or HIS-DTX3L-RING domain mutant (3C/A) were incubated with ATP, E1, E2 (UbcH5c), and ubiquitin in a final volume of 20 μl at room temperature for 1½ h. Reactions were stopped by the addition of 20 μl of 2× sample buffer. Proteins were resolved by 7% SDS–PAGE and analyzed by immunoblotting with the indicated antibodies. The C830A cleavage from GST was not complete, resulting in detection of multiple bands in both the input and reaction lanes (indicated with asterisks) that correspond to uncleaved and degraded products of GST-C830A, as determined by immunoblot analysis with an anti-GST antibody (unpublished data). A high–molecular weight nonspecific band is also noted with an asterisk. Data are representative of six independent experiments. (B) Purified AIP4, HIS-DTX3L, or MBP-Parkin were incubated with ATP, E1, E2 (UbcH5c), and ubiquitin in a final volume of 20 μl at room temperature for 1½ h. Reactions were stopped by the addition of 20 μl of 2× sample buffer. Proteins were resolved by 7% SDS–PAGE and analyzed by immunoblotting with the indicated antibodies. Data are representative of four independent experiments. (C) Hyperubiquitination of HRS in DTX3L-depleted cells is suppressed by AIP4 depletion. HeLa cells were transfected with siRNA directed against control, DTX3L, or AIP4 plus FLAG-ubiquitin and T7-HRS. Samples were processed as described in the legend to Figure 5, A and B. Data are representative of four independent experiments.

Article Snippet: The mouse monoclonal antibodies directed against HRS (c-19) and ubiquitin (P4D1) and the goat polyclonal directed against DTX3L (N-16) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Ubiquitin Proteomics, Activity Assay, Purification, Mutagenesis, Incubation, SDS Page, Western Blot, High Molecular Weight, Transfection, Control

Journal: BMC Plant Biology

Article Title: Selenite activates the alternative oxidase pathway and alters primary metabolism in Brassica napus roots: evidence of a mitochondrial stress response

doi: 10.1186/s12870-014-0259-6

Figure Lengend Snippet: The effects of selenite on the ubiquitin-proteasome pathway in root tissue. (a) Proteasome activity in roots of untreated plants and plants treated with 50 μM selenite for 1 and 3 d. Shown are the mean and SE in 5 different plants. Values represent fluorescence of proteasomally-released AMC at the each time interval. Data are the mean of three biological replicates and standard deviation. Lowercase letters represent a significant difference in activity at each time point ( p < 0.05). (b) The accumulation of high-molecular weight ubiquitinated proteins in the roots of B. napus from untreated plants and plants treated with 50 μM selenite for 1 and 3 days, and then supplemented in 0.1% DMSO with or without 100 μM MG132 in for 8 hours. 50 μg of protein were separated on an 8% SDS gel, and ubiquitinated proteins were detected using anti-ubiquitin antiserum. The immunoblot is representative of at least three biological experiments, and numbers below each blot represent the mean pixel intensity of all the immunoreactive bands relative to control on day 1. Asterisks indicate a significant difference in band intensity in selenite-treated plants compared to untreated plants (p < 0.05). L = ladder.

Article Snippet: Ubiquitinated proteins were detected on 8% gels containing 50 μg of protein per lane, which reacted against ubiquitin antiserum (Santa Cruz Biotechnology) as previously described [ ].

Techniques: Ubiquitin Proteomics, Activity Assay, Fluorescence, Standard Deviation, High Molecular Weight, SDS-Gel, Western Blot, Control