flag lysis buffer  (Roche)


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    Structured Review

    Roche flag lysis buffer
    In vitro RNA binding activity of full-length HBV with mutant T3 and RT1 motifs (a) Accumulation of HBV P mutants. HBV P derivatives were expressed in transfected 293T cells, immunoprecipitated with <t>anti-FLAG</t> antibodies, resolved by SDS-PAGE, and detected by western blotting using the <t>M2</t> anti-FLAG antibody; the exposure of the left gel was shorter to limit saturation of the more intense bands. The position of HBV P (P) and the antibody heavy chain (HC) are indicated. * denotes the position of an N-terminal fragment of the 3xFLAG-tagged wild-type P. (b) The immunoaffinity-purified HBV P derivatives were incubated with 32 P-labeled wild-type Hε or mutant Hε-dB RNA and co-precipitated products were resolved by SDS–PAGE. Input representing 0.5% of the indicated ε RNA added to each binding reaction mixture is in lanes 10, 11, 20, and 21. (c) Bound 32 P-labeled ε RNA signals were quantified via phosphorimaging and compared to the binding of wild-type P to Hε RNA. The data represent the mean ± one standard deviation from at least three independent experiments.
    Flag Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 89/100, based on 2781 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Sequences in the terminal protein and reverse transcriptase domains of the Hepatitis B Virus polymerase contribute to RNA binding and encapsidation"

    Article Title: Sequences in the terminal protein and reverse transcriptase domains of the Hepatitis B Virus polymerase contribute to RNA binding and encapsidation

    Journal: Journal of viral hepatitis

    doi: 10.1111/jvh.12225

    In vitro RNA binding activity of full-length HBV with mutant T3 and RT1 motifs (a) Accumulation of HBV P mutants. HBV P derivatives were expressed in transfected 293T cells, immunoprecipitated with anti-FLAG antibodies, resolved by SDS-PAGE, and detected by western blotting using the M2 anti-FLAG antibody; the exposure of the left gel was shorter to limit saturation of the more intense bands. The position of HBV P (P) and the antibody heavy chain (HC) are indicated. * denotes the position of an N-terminal fragment of the 3xFLAG-tagged wild-type P. (b) The immunoaffinity-purified HBV P derivatives were incubated with 32 P-labeled wild-type Hε or mutant Hε-dB RNA and co-precipitated products were resolved by SDS–PAGE. Input representing 0.5% of the indicated ε RNA added to each binding reaction mixture is in lanes 10, 11, 20, and 21. (c) Bound 32 P-labeled ε RNA signals were quantified via phosphorimaging and compared to the binding of wild-type P to Hε RNA. The data represent the mean ± one standard deviation from at least three independent experiments.
    Figure Legend Snippet: In vitro RNA binding activity of full-length HBV with mutant T3 and RT1 motifs (a) Accumulation of HBV P mutants. HBV P derivatives were expressed in transfected 293T cells, immunoprecipitated with anti-FLAG antibodies, resolved by SDS-PAGE, and detected by western blotting using the M2 anti-FLAG antibody; the exposure of the left gel was shorter to limit saturation of the more intense bands. The position of HBV P (P) and the antibody heavy chain (HC) are indicated. * denotes the position of an N-terminal fragment of the 3xFLAG-tagged wild-type P. (b) The immunoaffinity-purified HBV P derivatives were incubated with 32 P-labeled wild-type Hε or mutant Hε-dB RNA and co-precipitated products were resolved by SDS–PAGE. Input representing 0.5% of the indicated ε RNA added to each binding reaction mixture is in lanes 10, 11, 20, and 21. (c) Bound 32 P-labeled ε RNA signals were quantified via phosphorimaging and compared to the binding of wild-type P to Hε RNA. The data represent the mean ± one standard deviation from at least three independent experiments.

    Techniques Used: In Vitro, RNA Binding Assay, Activity Assay, Mutagenesis, Transfection, Immunoprecipitation, SDS Page, Western Blot, Purification, Incubation, Labeling, Binding Assay, Standard Deviation

    2) Product Images from "Mer receptor tyrosine kinase is a novel therapeutic target in pediatric B-cell acute lymphoblastic leukemia"

    Article Title: Mer receptor tyrosine kinase is a novel therapeutic target in pediatric B-cell acute lymphoblastic leukemia

    Journal: Blood

    doi: 10.1182/blood-2009-03-209247

    Abrogation of Mer expression in the E2A-PBX1 + human B-ALL cell line 697. Cells were infected with lentiviral particles containing short hairpin RNA (shRNA) constructs targeting Mer (shMer1A, shMer1B) or GFP (shControl) as a nonsilencing control. Mer knockdown
    Figure Legend Snippet: Abrogation of Mer expression in the E2A-PBX1 + human B-ALL cell line 697. Cells were infected with lentiviral particles containing short hairpin RNA (shRNA) constructs targeting Mer (shMer1A, shMer1B) or GFP (shControl) as a nonsilencing control. Mer knockdown

    Techniques Used: Expressing, Infection, shRNA, Construct

    3) Product Images from "Characterization of a core fragment of the rhesus monkey TRIM5? protein"

    Article Title: Characterization of a core fragment of the rhesus monkey TRIM5? protein

    Journal: BMC Biochemistry

    doi: 10.1186/1471-2091-12-1

    Oligomerization state of BCCL2 variants in mammalian cells . A . Lysates from 293 T cells expressing the wild-type (wt) and mutant BCCL2 proteins with V5 epitope tags were analyzed by 12% SDS-PAGE and Western blotted with an HRP-conjugated anti-V5 antibody. B . Lysates from 293 T cells expressing the wild-type (wt) and mutant BCCL2 proteins were treated with the indicated concentrations of glutaraldehyde and then boiled in Laemmli buffer and analyzed by SDS-PAGE and Western blotting, as described above. The positions of the molecular-weight markers in kD are shown. The arrows indicate the positions of monomers (M), dimers (D) and higher-order oligomers (H-O).
    Figure Legend Snippet: Oligomerization state of BCCL2 variants in mammalian cells . A . Lysates from 293 T cells expressing the wild-type (wt) and mutant BCCL2 proteins with V5 epitope tags were analyzed by 12% SDS-PAGE and Western blotted with an HRP-conjugated anti-V5 antibody. B . Lysates from 293 T cells expressing the wild-type (wt) and mutant BCCL2 proteins were treated with the indicated concentrations of glutaraldehyde and then boiled in Laemmli buffer and analyzed by SDS-PAGE and Western blotting, as described above. The positions of the molecular-weight markers in kD are shown. The arrows indicate the positions of monomers (M), dimers (D) and higher-order oligomers (H-O).

    Techniques Used: Expressing, Mutagenesis, SDS Page, Western Blot, Molecular Weight

    Electron microscopy of the LLER protein . A . The BCCL2 LLER protein purified by nickel-affinity, anion-exchange, and gel-filtration chromatography was applied to glow-discharged carbon grids. After staining with 1% uranyl formate, the grids were examined with a Tecnai G2 Spirit BioTWIN electron microscope (FEI Company) at 100 kV. B . The cryoelectron microscopic images of the LLER protein were taken at a magnification of 150,000 × and at a defocus of 3~5 μm with a Tecnai F20 field-emission gun electron microscope operating at 200 kV. The proteins that were purified as described above were embedded in a thin ice film on a Quantifoil grid, using an FEI Vitrobot, a robot that swiftly plunges the protein-loaded grid into liquid ethane. The images were low-pass filtered with background noises removed (right column). The bars in the left-hand images are 20 nm. C . The Peak 2 fraction of the LLER protein was incubated with an anti-His 6 antibody and imaged by single-particle cryoelectron microscopy, as described above. Representative images of the LLER protein alone (panel 1), the antibody alone (panel 2), and the LLER protein complexed with one or two antibody molecules (panels 3 and 4, respectively) are shown.
    Figure Legend Snippet: Electron microscopy of the LLER protein . A . The BCCL2 LLER protein purified by nickel-affinity, anion-exchange, and gel-filtration chromatography was applied to glow-discharged carbon grids. After staining with 1% uranyl formate, the grids were examined with a Tecnai G2 Spirit BioTWIN electron microscope (FEI Company) at 100 kV. B . The cryoelectron microscopic images of the LLER protein were taken at a magnification of 150,000 × and at a defocus of 3~5 μm with a Tecnai F20 field-emission gun electron microscope operating at 200 kV. The proteins that were purified as described above were embedded in a thin ice film on a Quantifoil grid, using an FEI Vitrobot, a robot that swiftly plunges the protein-loaded grid into liquid ethane. The images were low-pass filtered with background noises removed (right column). The bars in the left-hand images are 20 nm. C . The Peak 2 fraction of the LLER protein was incubated with an anti-His 6 antibody and imaged by single-particle cryoelectron microscopy, as described above. Representative images of the LLER protein alone (panel 1), the antibody alone (panel 2), and the LLER protein complexed with one or two antibody molecules (panels 3 and 4, respectively) are shown.

    Techniques Used: Electron Microscopy, Purification, Filtration, Chromatography, Staining, Microscopy, Incubation

    Secondary structure and melting temperature of the LLER protein . A . The far-UV spectra (195 - 245 nm) of the LLER protein were recorded with an Aviv circular dichroism (CD) spectrometer at the indicated temperatures. The percentage of alpha-helical content at the various temperatures was calculated and is shown in the key. B . The melting curve of the BCCL2 protein was generated by plotting the alpha-helical content as a function of temperature. Note the biphasic shape of the curve. C . Approximately 2 μg of the BCCL2 protein was incubated at the indicated temperatures for 5 minutes prior to the addition of 1 mM glutaraldehyde. Incubation at the same temperature was continued for another 8 minutes, after which the reaction was quenched by addition of excess 0.1 M Tris-HCl, pH 7.5 buffer. A control reaction without the addition of glutaraldehyde was also performed at each temperature. The reaction mixtures were boiled in Laemmli buffer and analyzed on a 4-12% SDS-polyacrylamide gel. M, molecular weight markers.
    Figure Legend Snippet: Secondary structure and melting temperature of the LLER protein . A . The far-UV spectra (195 - 245 nm) of the LLER protein were recorded with an Aviv circular dichroism (CD) spectrometer at the indicated temperatures. The percentage of alpha-helical content at the various temperatures was calculated and is shown in the key. B . The melting curve of the BCCL2 protein was generated by plotting the alpha-helical content as a function of temperature. Note the biphasic shape of the curve. C . Approximately 2 μg of the BCCL2 protein was incubated at the indicated temperatures for 5 minutes prior to the addition of 1 mM glutaraldehyde. Incubation at the same temperature was continued for another 8 minutes, after which the reaction was quenched by addition of excess 0.1 M Tris-HCl, pH 7.5 buffer. A control reaction without the addition of glutaraldehyde was also performed at each temperature. The reaction mixtures were boiled in Laemmli buffer and analyzed on a 4-12% SDS-polyacrylamide gel. M, molecular weight markers.

    Techniques Used: Generated, Incubation, Molecular Weight

    Effect of B-box 2 changes on BCCL2 expression and solubility . The wild-type (wt) BCCL2 protein and the indicated B-box 2 mutants were expressed in E. coli. The bacteria were lysed and the lysates centrifuged at 4000 × g for 10 minutes. The supernatants were loaded onto a Ni +2 -NTA affinity column; the proteins eluted with 300 mM imidazole were analyzed by SDS-PAGE and Coomassie Blue staining.
    Figure Legend Snippet: Effect of B-box 2 changes on BCCL2 expression and solubility . The wild-type (wt) BCCL2 protein and the indicated B-box 2 mutants were expressed in E. coli. The bacteria were lysed and the lysates centrifuged at 4000 × g for 10 minutes. The supernatants were loaded onto a Ni +2 -NTA affinity column; the proteins eluted with 300 mM imidazole were analyzed by SDS-PAGE and Coomassie Blue staining.

    Techniques Used: Expressing, Solubility, Affinity Column, SDS Page, Staining

    Comparison of the size-exclusion chromatography profiles of the wild-type and mutant BCCL2 proteins . A . Purified wild-type (wt) and mutant BCCL2 proteins were loaded onto a gel-filtration column and eluted at a flow rate of 0.3 ml/min. The protein peaks 1 (P1) and 2 (P2) are indicated. The positions at which the globular proteins standards thyroglobulin (670 kD) and bovine gamma-globulin (158 kD) were eluted in a parallel run are indicated. B . Fractions from the gel-filtration column were separated on a 12% SDS-polyacrylamide gel, which was stained with Coomassie Blue (bottom panel). The 25- and 37-kD molecular weight markers (M) are shown in the left-most lanes. An aliquot of the LLWL protein loaded on the gel-filtration column was also analyzed (Load). The positions of peaks P1 and P2 are noted.
    Figure Legend Snippet: Comparison of the size-exclusion chromatography profiles of the wild-type and mutant BCCL2 proteins . A . Purified wild-type (wt) and mutant BCCL2 proteins were loaded onto a gel-filtration column and eluted at a flow rate of 0.3 ml/min. The protein peaks 1 (P1) and 2 (P2) are indicated. The positions at which the globular proteins standards thyroglobulin (670 kD) and bovine gamma-globulin (158 kD) were eluted in a parallel run are indicated. B . Fractions from the gel-filtration column were separated on a 12% SDS-polyacrylamide gel, which was stained with Coomassie Blue (bottom panel). The 25- and 37-kD molecular weight markers (M) are shown in the left-most lanes. An aliquot of the LLWL protein loaded on the gel-filtration column was also analyzed (Load). The positions of peaks P1 and P2 are noted.

    Techniques Used: Size-exclusion Chromatography, Mutagenesis, Purification, Filtration, Flow Cytometry, Staining, Molecular Weight

    Purification of the BCCL2 protein expressed in bacteria . A . Bacterial cells expressing the BCCL2 protein were lysed and the homogenates subjected to purification approaches. In lane 1, the soluble BCCL2 protein was purified by Ni +2 -NTA metal-affinity chromatography. Lane 2 shows the insoluble pellet obtained after lysis of the bacteria with lysis buffer. The proteins in each sample were resolved by SDS-PAGE and Coomassie Blue staining. B . The affinity-purified BCCL2 protein was loaded onto a gel-filtration column and eluted at a flow rate of 0.3 ml/min. The OD 280 of the eluted protein is plotted (blue line). The profile of the globular protein standards (thyroglobulin (670 kD), bovine gamma-globulin (158 kD), chicken ovalbumin (44 kD), equine myoglobin (17 kD) and vitamin B12 (1.35 kD) is shown in red. Fractions from the gel-filtration column were separated on a 12% SDS-polyacrylamide gel, which was stained with Coomassie Blue. An aliquot of the BCCL2 protein sample loaded on the gel-filtration column was analyzed (Load), along with the molecular-weight markers (M). C . The affinity-purified BCCL2 protein was loaded onto a Hi-trap Q anion-exchange column and eluted at a flow rate of 0.5 ml/min (left panel). The fractions from the column were separated on a 12% SDS-polyacrylamide gel, which was stained with Coomassie Blue (right panel).
    Figure Legend Snippet: Purification of the BCCL2 protein expressed in bacteria . A . Bacterial cells expressing the BCCL2 protein were lysed and the homogenates subjected to purification approaches. In lane 1, the soluble BCCL2 protein was purified by Ni +2 -NTA metal-affinity chromatography. Lane 2 shows the insoluble pellet obtained after lysis of the bacteria with lysis buffer. The proteins in each sample were resolved by SDS-PAGE and Coomassie Blue staining. B . The affinity-purified BCCL2 protein was loaded onto a gel-filtration column and eluted at a flow rate of 0.3 ml/min. The OD 280 of the eluted protein is plotted (blue line). The profile of the globular protein standards (thyroglobulin (670 kD), bovine gamma-globulin (158 kD), chicken ovalbumin (44 kD), equine myoglobin (17 kD) and vitamin B12 (1.35 kD) is shown in red. Fractions from the gel-filtration column were separated on a 12% SDS-polyacrylamide gel, which was stained with Coomassie Blue. An aliquot of the BCCL2 protein sample loaded on the gel-filtration column was analyzed (Load), along with the molecular-weight markers (M). C . The affinity-purified BCCL2 protein was loaded onto a Hi-trap Q anion-exchange column and eluted at a flow rate of 0.5 ml/min (left panel). The fractions from the column were separated on a 12% SDS-polyacrylamide gel, which was stained with Coomassie Blue (right panel).

    Techniques Used: Purification, Expressing, Affinity Chromatography, Lysis, SDS Page, Staining, Affinity Purification, Filtration, Flow Cytometry, Molecular Weight

    4) Product Images from "Rational design of antisense oligonucleotides targeting single nucleotide polymorphisms for potent and allele selective suppression of mutant Huntingtin in the CNS"

    Article Title: Rational design of antisense oligonucleotides targeting single nucleotide polymorphisms for potent and allele selective suppression of mutant Huntingtin in the CNS

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt725

    Activity and selectivity of ASOs in the rodent CNS. ( A ) Allele selective knockdown of muHTT protein with ASO A30 in neuronal cells derived from cortical and striatal tissues of Hu97/18 mouse embryos under free-uptake conditions. ( B–D ) Hu97/18 mice ( n = 4/group) were injected ICV with a single dose of 300 µg of ASO (B and D) or the indicated dose (C) in PBS. Mice were sacrificed after 28 days, the brain was harvested, and a 2 mm coronal slab from each hemisphere (R,L) was analyzed by allelic separation immunoblotting for muHTT and wtHTT protein, and the results were normalized to calnexin protein. (B) Optimized ASOs A25 , A26 , A30 and A31 show similar activity but improved allele selectivity relative to control ASO A1 . (C) Dose response for allele-selective knockdown of muHTT protein following ICV injection of ASO A30 in Hu97/18 mice (D) Immunohistochemical staining for ASO (red) illustrates distribution to al l parts of the brain following a single ICV bolus injection.
    Figure Legend Snippet: Activity and selectivity of ASOs in the rodent CNS. ( A ) Allele selective knockdown of muHTT protein with ASO A30 in neuronal cells derived from cortical and striatal tissues of Hu97/18 mouse embryos under free-uptake conditions. ( B–D ) Hu97/18 mice ( n = 4/group) were injected ICV with a single dose of 300 µg of ASO (B and D) or the indicated dose (C) in PBS. Mice were sacrificed after 28 days, the brain was harvested, and a 2 mm coronal slab from each hemisphere (R,L) was analyzed by allelic separation immunoblotting for muHTT and wtHTT protein, and the results were normalized to calnexin protein. (B) Optimized ASOs A25 , A26 , A30 and A31 show similar activity but improved allele selectivity relative to control ASO A1 . (C) Dose response for allele-selective knockdown of muHTT protein following ICV injection of ASO A30 in Hu97/18 mice (D) Immunohistochemical staining for ASO (red) illustrates distribution to al l parts of the brain following a single ICV bolus injection.

    Techniques Used: Activity Assay, Allele-specific Oligonucleotide, Derivative Assay, Mouse Assay, Injection, Immunohistochemistry, Staining

    5) Product Images from "Positive and Negative Regulation of Vertebrate Separase by Cdk1-Cyclin B1 May Explain Why Securin Is Dispensable *"

    Article Title: Positive and Negative Regulation of Vertebrate Separase by Cdk1-Cyclin B1 May Explain Why Securin Is Dispensable *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.615310

    Mitosis-specific phosphorylation of Ser-1126 decreases the solubility of separase. A , aggravated aggregation tendency of separase ( Sep. ) in mitosis. Synchronized interphase ( Int ., Interph. ) or prometaphase ( Mit .) transgenic HEK293 cells induced to express
    Figure Legend Snippet: Mitosis-specific phosphorylation of Ser-1126 decreases the solubility of separase. A , aggravated aggregation tendency of separase ( Sep. ) in mitosis. Synchronized interphase ( Int ., Interph. ) or prometaphase ( Mit .) transgenic HEK293 cells induced to express

    Techniques Used: Solubility, Transgenic Assay

    Positive and negative effects of Cdk1-cyclin B1 define the minimal requirements of separase regulation in mitosis. A , WT separase and mutants S1126A and ΔCLD exhibit some, high, or no cohesin cleavage activity upon isolation from prometaphase-arrested
    Figure Legend Snippet: Positive and negative effects of Cdk1-cyclin B1 define the minimal requirements of separase regulation in mitosis. A , WT separase and mutants S1126A and ΔCLD exhibit some, high, or no cohesin cleavage activity upon isolation from prometaphase-arrested

    Techniques Used: Activity Assay, Isolation

    6) Product Images from "Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Non-redundant Functions in Vivo"

    Article Title: Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Non-redundant Functions in Vivo

    Journal: Current Biology

    doi: 10.1016/j.cub.2018.10.002

    Contribution of Distinct Rac Binding Sites in Sra-1 to Lamellipodia Formation (A) Cell morphologies and lamellipodial phenotypes of B16-F1 control versus Sra-1/PIR121 KO cells (clone 3) transfected with EGFP or EGFP-tagged Sra-1, and stained for the actin cytoskeleton with phalloidin (scale bars, 20 μm). (B) Cell lysates of B16-F1 cells, Sra-1/PIR121 KO cells (clone 3), as well as KO cells expressing EGFP-Sra-1 were subjected to western blotting to detect expression levels of WAVE complex components, as indicated. (C) B16-F1 control cells, Sra-1/PIR121 KO clone 3, and the latter forming lamellipodia upon transfection with EGFP-tagged Sra-1 were analyzed for random migration speed ( ∗∗∗ p ≤ 0.001; n.s. [not significant]: p > 0.05). Box and whisker plots represent data as follows: boxes correspond to 50% of data points (25%–75%), and whiskers correspond to 80% (10%–90%). Outliers are shown as dots, and lines and red numbers in boxes correspond to medians. ]). From the view chosen, only WAVE (magenta), Sra-1 (green), and Nap1 (blue) are visible. Sra-1 possesses two binding sites for Rac (termed A site and D site) and sequesters the WH2 and C regions of WAVE. Rac binding to Sra-1 is thought to release interactions with the WH2 and C regions, thereby activating the WCA domain of WAVE. (E) Sra-1/PIR121 KO cells (clone 3) were transfected with EGFP or various EGFP-Sra-1 constructs, lysed, and subjected to pull-downs with constitutively active Rac1 (Rac1-L61). Note strongly increased interaction of the WCA ∗ mutant with Rac1, which was strongly and virtually entirely diminished upon additional mutation of the A and D site, respectively. Combinatorial mutation of both Rac binding sites in the WCA ∗ background appeared to abolish detectable Rac1 interaction entirely. WCA ∗ : disrupted WH2 and C region contact sites (L697D/Y704D/L841A/F844A/W845A). (F) Sra-1/PIR121 KO cells (clone 3) were transfected with the indicated EGFP-Sra-1 constructs and assayed for lamellipodia formation. Lamellipodial actin networks that were small, narrow, or displayed multiple ruffles were defined as “immature lamellipodia,” marked by arrowheads in cell images (right), as opposed to regular lamellipodia, marked by arrows (scale bar, 10 μm). Data in the bar chart are arithmetic means ± SEM from three independent experiments. Note that the A site mutation diminished lamellipodia formation in a fashion that could be restored by additional WCA ∗ mutation of Sra-1. In the case of the D site, lamellipodial morphology was compromised in a fashion mostly independent from the WCA ∗ mutation. The WIRS mutation had no detectable effect. To assess statistical significance of differences or confirm the absence of statistically relevant differences between experimental groups, a non-parametric, Mann-Whitney rank-sum test was performed in multiple, individual combinations of datasets. For each experimental group, we compared the number of cells with regular, i.e., “fully developed” lamellipodia, immature lamellipodia (see above), or the two groups combined, and hence all cells display either one of the lamellipodium-like structures. Selected combinations are as follows, with three p values representing aforementioned lamellipodial categories: WT-WIRS (n.s., n.s., n.s.); WT-C179R/R190D+WCA ∗ (n.s., n.s., n.s.); WT-Y967A ( ∗∗ , ∗∗ , n.s.); WT-G971W ( ∗∗ , ∗ , n.s.); WT-Y967A+WCA ∗ ( ∗∗ , ∗∗ , n.s.); Y967A-Y967A+WCA ∗ ( ∗ , n.s., n.s.); WT-WCA ∗ (n.s., n.s., ∗∗ ). Statistical significance is expressed as ∗∗ p ≤ 0.01, ∗ p ≤ 0.05, and n.s. (not significant): p > 0.05. WIRS: Y923A/E1084A to mutate the WIRS-binding pocket; WCA ∗ : disrupted WH2 and C region contact sites (L697D/Y704D/L841A/F844A/W845A). .
    Figure Legend Snippet: Contribution of Distinct Rac Binding Sites in Sra-1 to Lamellipodia Formation (A) Cell morphologies and lamellipodial phenotypes of B16-F1 control versus Sra-1/PIR121 KO cells (clone 3) transfected with EGFP or EGFP-tagged Sra-1, and stained for the actin cytoskeleton with phalloidin (scale bars, 20 μm). (B) Cell lysates of B16-F1 cells, Sra-1/PIR121 KO cells (clone 3), as well as KO cells expressing EGFP-Sra-1 were subjected to western blotting to detect expression levels of WAVE complex components, as indicated. (C) B16-F1 control cells, Sra-1/PIR121 KO clone 3, and the latter forming lamellipodia upon transfection with EGFP-tagged Sra-1 were analyzed for random migration speed ( ∗∗∗ p ≤ 0.001; n.s. [not significant]: p > 0.05). Box and whisker plots represent data as follows: boxes correspond to 50% of data points (25%–75%), and whiskers correspond to 80% (10%–90%). Outliers are shown as dots, and lines and red numbers in boxes correspond to medians. ]). From the view chosen, only WAVE (magenta), Sra-1 (green), and Nap1 (blue) are visible. Sra-1 possesses two binding sites for Rac (termed A site and D site) and sequesters the WH2 and C regions of WAVE. Rac binding to Sra-1 is thought to release interactions with the WH2 and C regions, thereby activating the WCA domain of WAVE. (E) Sra-1/PIR121 KO cells (clone 3) were transfected with EGFP or various EGFP-Sra-1 constructs, lysed, and subjected to pull-downs with constitutively active Rac1 (Rac1-L61). Note strongly increased interaction of the WCA ∗ mutant with Rac1, which was strongly and virtually entirely diminished upon additional mutation of the A and D site, respectively. Combinatorial mutation of both Rac binding sites in the WCA ∗ background appeared to abolish detectable Rac1 interaction entirely. WCA ∗ : disrupted WH2 and C region contact sites (L697D/Y704D/L841A/F844A/W845A). (F) Sra-1/PIR121 KO cells (clone 3) were transfected with the indicated EGFP-Sra-1 constructs and assayed for lamellipodia formation. Lamellipodial actin networks that were small, narrow, or displayed multiple ruffles were defined as “immature lamellipodia,” marked by arrowheads in cell images (right), as opposed to regular lamellipodia, marked by arrows (scale bar, 10 μm). Data in the bar chart are arithmetic means ± SEM from three independent experiments. Note that the A site mutation diminished lamellipodia formation in a fashion that could be restored by additional WCA ∗ mutation of Sra-1. In the case of the D site, lamellipodial morphology was compromised in a fashion mostly independent from the WCA ∗ mutation. The WIRS mutation had no detectable effect. To assess statistical significance of differences or confirm the absence of statistically relevant differences between experimental groups, a non-parametric, Mann-Whitney rank-sum test was performed in multiple, individual combinations of datasets. For each experimental group, we compared the number of cells with regular, i.e., “fully developed” lamellipodia, immature lamellipodia (see above), or the two groups combined, and hence all cells display either one of the lamellipodium-like structures. Selected combinations are as follows, with three p values representing aforementioned lamellipodial categories: WT-WIRS (n.s., n.s., n.s.); WT-C179R/R190D+WCA ∗ (n.s., n.s., n.s.); WT-Y967A ( ∗∗ , ∗∗ , n.s.); WT-G971W ( ∗∗ , ∗ , n.s.); WT-Y967A+WCA ∗ ( ∗∗ , ∗∗ , n.s.); Y967A-Y967A+WCA ∗ ( ∗ , n.s., n.s.); WT-WCA ∗ (n.s., n.s., ∗∗ ). Statistical significance is expressed as ∗∗ p ≤ 0.01, ∗ p ≤ 0.05, and n.s. (not significant): p > 0.05. WIRS: Y923A/E1084A to mutate the WIRS-binding pocket; WCA ∗ : disrupted WH2 and C region contact sites (L697D/Y704D/L841A/F844A/W845A). .

    Techniques Used: Binding Assay, Transfection, Staining, Expressing, Western Blot, Migration, Whisker Assay, Construct, Mutagenesis, MANN-WHITNEY

    7) Product Images from "Alternative Intronic Polyadenylation Generates the Interleukin-6 Trans-signaling Inhibitor sgp130-E10 *"

    Article Title: Alternative Intronic Polyadenylation Generates the Interleukin-6 Trans-signaling Inhibitor sgp130-E10 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.560938

    sgp130-E10 is conserved in many mammals and most abundantly expressed in blood cells. A , schematic overview of gp130 and the novel sgp130-E10 variant in comparison with the three transcripts formed by alternative polyadenylation. Exons are indicated by
    Figure Legend Snippet: sgp130-E10 is conserved in many mammals and most abundantly expressed in blood cells. A , schematic overview of gp130 and the novel sgp130-E10 variant in comparison with the three transcripts formed by alternative polyadenylation. Exons are indicated by

    Techniques Used: Variant Assay

    Analysis of the binding of Hyper-IL-6 to captured sgp130Fc and gp130-E10Fc by surface plasmon resonance spectroscopy. A , sgp130-E10Fc and B , sgp130Fc were captured to immobilized human Fc antibody, and Hyper-IL-6 was injected for 180 s at concentrations
    Figure Legend Snippet: Analysis of the binding of Hyper-IL-6 to captured sgp130Fc and gp130-E10Fc by surface plasmon resonance spectroscopy. A , sgp130-E10Fc and B , sgp130Fc were captured to immobilized human Fc antibody, and Hyper-IL-6 was injected for 180 s at concentrations

    Techniques Used: Binding Assay, SPR Assay, Spectroscopy, Injection

    Specific detection of sgp130-E10 by the monoclonal sgp130-E10 antibody E10/1 in PBMCs and serum. A , Western blot ( WB ) analysis of sgp130-E10Fc and sgp130Fc in different concentrations. The monoclonal sgp130-E10 antibody E10/1 specifically detected recombinant
    Figure Legend Snippet: Specific detection of sgp130-E10 by the monoclonal sgp130-E10 antibody E10/1 in PBMCs and serum. A , Western blot ( WB ) analysis of sgp130-E10Fc and sgp130Fc in different concentrations. The monoclonal sgp130-E10 antibody E10/1 specifically detected recombinant

    Techniques Used: Western Blot, Recombinant

    sgp130-E10Fc interacts with Hyper-IL-6, but not with IL-6 alone. A , Coomassie Blue stains of sgp130-E10Fc and sgp130Fc. Different amounts from 1 to 20 μg of sgp130-E10Fc and sgp130Fc were separated by SDS-PAGE and visualized by Coomassie Blue
    Figure Legend Snippet: sgp130-E10Fc interacts with Hyper-IL-6, but not with IL-6 alone. A , Coomassie Blue stains of sgp130-E10Fc and sgp130Fc. Different amounts from 1 to 20 μg of sgp130-E10Fc and sgp130Fc were separated by SDS-PAGE and visualized by Coomassie Blue

    Techniques Used: SDS Page

    Structure of sgp130-E10 and its interaction with Hyper-IL-6. A , schematic overview of sgp130-E10 in complex with IL-6/IL-6R. B , immunoprecipitation with conditioned medium from HEK-293 cell cultures transiently transfected with plasmids coding for sgp130-E10Myc-His
    Figure Legend Snippet: Structure of sgp130-E10 and its interaction with Hyper-IL-6. A , schematic overview of sgp130-E10 in complex with IL-6/IL-6R. B , immunoprecipitation with conditioned medium from HEK-293 cell cultures transiently transfected with plasmids coding for sgp130-E10Myc-His

    Techniques Used: Immunoprecipitation, Transfection

    Purified sgp130-E10Fc is biologically active, but inferior to sgp130Fc in terms of binding to Hyper-IL-6, activity and stability. A , competitive ELISA with coated sgp130Fc and competition with either sgp130Fc or sgp130-E10Fc for Hyper-IL-6 binding. Standard
    Figure Legend Snippet: Purified sgp130-E10Fc is biologically active, but inferior to sgp130Fc in terms of binding to Hyper-IL-6, activity and stability. A , competitive ELISA with coated sgp130Fc and competition with either sgp130Fc or sgp130-E10Fc for Hyper-IL-6 binding. Standard

    Techniques Used: Purification, Binding Assay, Activity Assay, Competitive ELISA

    sgp130-E10 protein Binds to IL-6/Soluble IL-6R (Hyper-IL-6), but Not to IL-6 Alone
    Figure Legend Snippet: sgp130-E10 protein Binds to IL-6/Soluble IL-6R (Hyper-IL-6), but Not to IL-6 Alone

    Techniques Used:

    8) Product Images from "EMT Reversal in human cancer cells after IR knockdown in hyperinsulinemic mice"

    Article Title: EMT Reversal in human cancer cells after IR knockdown in hyperinsulinemic mice

    Journal: Endocrine-related cancer

    doi: 10.1530/ERC-16-0142

    Insulin Receptor Knockdown inhibited pulmonary metastases in hyperinsulinemic mice. Lungs were fixed, paraffin embedded and sectioned. Representative images of H E stained lung sections from Rag/WT and Rag/MKR mice injected with LCC6 Ctrl and
    Figure Legend Snippet: Insulin Receptor Knockdown inhibited pulmonary metastases in hyperinsulinemic mice. Lungs were fixed, paraffin embedded and sectioned. Representative images of H E stained lung sections from Rag/WT and Rag/MKR mice injected with LCC6 Ctrl and

    Techniques Used: Mouse Assay, Staining, Injection

    Silencing the insulin receptor leads to decreased tumor growth. 8–10 week old Rag/WT control and Rag/MKR hyperinsulinemic mice were injected with either 5×10 6 LCC6 control (Ctrl) or 5×10 6 LCC6 insulin receptor knockdown (IRKD)
    Figure Legend Snippet: Silencing the insulin receptor leads to decreased tumor growth. 8–10 week old Rag/WT control and Rag/MKR hyperinsulinemic mice were injected with either 5×10 6 LCC6 control (Ctrl) or 5×10 6 LCC6 insulin receptor knockdown (IRKD)

    Techniques Used: Mouse Assay, Injection

    Expression of IR and IGF1R in LCC6 Tumors. (A) Primary tumors from Rag/WT and Rag/MKR mice were assessed for the gene expression of the insulin receptor, demonstrating an 83% reduction of IR in the tumors from the LCC6 IRKD cells. (* p
    Figure Legend Snippet: Expression of IR and IGF1R in LCC6 Tumors. (A) Primary tumors from Rag/WT and Rag/MKR mice were assessed for the gene expression of the insulin receptor, demonstrating an 83% reduction of IR in the tumors from the LCC6 IRKD cells. (* p

    Techniques Used: Expressing, Mouse Assay

    Reduction of insulin signaling pathway in LCC6 IRKD tumors. (A) Representative blots showing protein extracted from tumor tissue and analyzed by Western blot for phospho-Akt (pAKT) and total AKT expression. B-Actin antibody used as loading control. Densitometry
    Figure Legend Snippet: Reduction of insulin signaling pathway in LCC6 IRKD tumors. (A) Representative blots showing protein extracted from tumor tissue and analyzed by Western blot for phospho-Akt (pAKT) and total AKT expression. B-Actin antibody used as loading control. Densitometry

    Techniques Used: Western Blot, Expressing

    Tumors from LCC6 IRKD cells have reversal of Epithelial-Mesenchymal Transition phenotype. (A) Representative blots showing protein extracted from tumor tissue and analyzed by Western blot for Vimentin expression. B-Actin antibody was used as the loading
    Figure Legend Snippet: Tumors from LCC6 IRKD cells have reversal of Epithelial-Mesenchymal Transition phenotype. (A) Representative blots showing protein extracted from tumor tissue and analyzed by Western blot for Vimentin expression. B-Actin antibody was used as the loading

    Techniques Used: Western Blot, Expressing

    9) Product Images from "IscR Is a Global Regulator Essential for Pathogenesis of Vibrio vulnificus and Induced by Host Cells"

    Article Title: IscR Is a Global Regulator Essential for Pathogenesis of Vibrio vulnificus and Induced by Host Cells

    Journal: Infection and Immunity

    doi: 10.1128/IAI.01141-13

    IscR-regulated genes possibly involved in the pathogenesis of V. vulnificus . Twelve genes possibly involved in the pathogenesis of V. vulnificus were chosen from the pool of the IscR regulon members predicted by microarray analysis. Regulation of their
    Figure Legend Snippet: IscR-regulated genes possibly involved in the pathogenesis of V. vulnificus . Twelve genes possibly involved in the pathogenesis of V. vulnificus were chosen from the pool of the IscR regulon members predicted by microarray analysis. Regulation of their

    Techniques Used: Microarray

    Induction of iscR expression by INT-407 host cells. Wild-type V. vulnificus was exposed to various numbers of INT-407 cells for 30 min as indicated and then used to isolate total RNAs and proteins as described in Materials and Methods. (A) The iscR mRNA
    Figure Legend Snippet: Induction of iscR expression by INT-407 host cells. Wild-type V. vulnificus was exposed to various numbers of INT-407 cells for 30 min as indicated and then used to isolate total RNAs and proteins as described in Materials and Methods. (A) The iscR mRNA

    Techniques Used: Expressing

    Adhesion of the V. vulnificus strains. (A) INT-407 cells were infected at an MOI of 10 with the V. vulnificus strains as indicated. After 30 min, adherent bacteria were enumerated, and results are presented as the numbers of bacteria per well of the tissue
    Figure Legend Snippet: Adhesion of the V. vulnificus strains. (A) INT-407 cells were infected at an MOI of 10 with the V. vulnificus strains as indicated. After 30 min, adherent bacteria were enumerated, and results are presented as the numbers of bacteria per well of the tissue

    Techniques Used: Infection

    Effects of scavenging ROS on host cell induction of iscR expression. (A and B) Wild-type V. vulnificus grown to an A 600 of 0.5 was exposed to MEMF (control), 4 × 10 6 INT-407 cells, or 4 × 10 6 INT-407 cells preincubated with NAC for 30
    Figure Legend Snippet: Effects of scavenging ROS on host cell induction of iscR expression. (A and B) Wild-type V. vulnificus grown to an A 600 of 0.5 was exposed to MEMF (control), 4 × 10 6 INT-407 cells, or 4 × 10 6 INT-407 cells preincubated with NAC for 30

    Techniques Used: Expressing

    Motility of the V. vulnificus strains. (A) The areas of motility of the strains grown at 30°C for 24 h on plates with LBS and 0.3% agar were photographed. (B) The diameters of motility areas are the means plus SEM of results from three independent
    Figure Legend Snippet: Motility of the V. vulnificus strains. (A) The areas of motility of the strains grown at 30°C for 24 h on plates with LBS and 0.3% agar were photographed. (B) The diameters of motility areas are the means plus SEM of results from three independent

    Techniques Used:

    Cytotoxicity and mouse mortality of V. vulnificus . (A) INT-407 cells were infected with the V. vulnificus strains at an MOI of 10. The cytotoxicity was determined by an LDH release assay and expressed using the total LDH released from the cells completely
    Figure Legend Snippet: Cytotoxicity and mouse mortality of V. vulnificus . (A) INT-407 cells were infected with the V. vulnificus strains at an MOI of 10. The cytotoxicity was determined by an LDH release assay and expressed using the total LDH released from the cells completely

    Techniques Used: Infection, Lactate Dehydrogenase Assay

    Growth of V. vulnificus under oxidative stress. The V. vulnificus strains were compared for their ability to grow on LBS plates supplemented without oxidants (LBS) or with 250 μM H 2 O 2 or 60 μM t -BOOH. Serial 10-fold dilutions of each culture
    Figure Legend Snippet: Growth of V. vulnificus under oxidative stress. The V. vulnificus strains were compared for their ability to grow on LBS plates supplemented without oxidants (LBS) or with 250 μM H 2 O 2 or 60 μM t -BOOH. Serial 10-fold dilutions of each culture

    Techniques Used:

    Hemolytic activities of V. vulnificus . An aliquot of the culture supernatants of V. vulnificus strains was mixed with an equal volume of hRBCs and then incubated at 37°C for 20 min. The lysis of hRBCs was determined, and results are presented
    Figure Legend Snippet: Hemolytic activities of V. vulnificus . An aliquot of the culture supernatants of V. vulnificus strains was mixed with an equal volume of hRBCs and then incubated at 37°C for 20 min. The lysis of hRBCs was determined, and results are presented

    Techniques Used: Incubation, Lysis

    Induction of iscR expression by H 2 O 2 . Total RNAs and proteins were isolated from wild-type V. vulnificus , grown anaerobically to an A 600 of 0.5, and then exposed to various levels of H 2 O 2 for 10 min as indicated. (A) The iscR mRNA levels were determined
    Figure Legend Snippet: Induction of iscR expression by H 2 O 2 . Total RNAs and proteins were isolated from wild-type V. vulnificus , grown anaerobically to an A 600 of 0.5, and then exposed to various levels of H 2 O 2 for 10 min as indicated. (A) The iscR mRNA levels were determined

    Techniques Used: Expressing, Isolation

    10) Product Images from "The Plasmodium Class XIV Myosin, MyoB, Has a Distinct Subcellular Location in Invasive and Motile Stages of the Malaria Parasite and an Unusual Light Chain *"

    Article Title: The Plasmodium Class XIV Myosin, MyoB, Has a Distinct Subcellular Location in Invasive and Motile Stages of the Malaria Parasite and an Unusual Light Chain *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.637694

    Generation of PfMyoB-GFP and PfMyoA-GFP parasites. A, schematic representation of the GFP-tagging of PfMyoB by single crossover homologous recombination into the myoB locus. The primers for PCR ( arrows 1 and 2 ) and the Southern blot probe together with restriction sites are labeled. X = XbaI and H = HpaI. B, diagnostic PCR on genomic DNA showing integration of PfMyoB-GFP ( primers 3 + 5 ) and wild type ( primers 3 + 4 ). Two PfMyoBGFP clones were examined. C, Southern blot analysis of cloned PfMyoB-GFP parasites. Genomic DNA was digested with XbaI and HpaI restriction enzymes. A probe to the myob region of homology showed the following: PfMyoB-GFP cycle 0 ( c0 ) shows the presence of wild-type (8.4 kb) and episome (4.3 kb) bands; 3D7 parasites only show the wild-type band. Clone 1 shows the expected bands for integration (7.9 and 4.8 kb), but also for episome, suggesting concatamer insertion. Clone 2 shows only bands for integration and was therefore used in all subsequent experiments. D, Western blot. Extracts of late stage schizonts from 3D7 and PfMyoB-GFP clone 2 parasites were immunoblotted wth an anti-GFP antibody. MyoB-GFP protein of ∼120 kDa was detected in clone 2. E, schematic representation of the GFP tagging of MyoA by single crossover homologous recombination into the myoA locus, with primers for PCR ( arrows with primer pair 15 and 16) and Southern blot probe and restriction sites labeled. C = ClaI and B = BsrFI. F, diagnostic PCR on genomic DNA showing integration of PfMyoA-GFP ( primers 17 + 5 ) and wild type ( primers 17 + 18 ). Four PfMyoA-GFP-expressing clones were examined. G, PfMyoA-GFP-expressing merozoites as viewed by live fluorescence microscopy. GFP was detected by green fluorescence, and the nuclei ( blue ) were labeled with Hoechst dye prior to microscopic analysis; the GFP signal is distributed to the parasite periphery. Scale bar, 2 μm. H, Southern blot analysis of cloned PfMyoA-GFP-expressing parasites. Genomic DNA was digested with ClaI and BsrFI. When probed with the myoa region of homology, all clones showed the expected two integration bands at 11.1 and 2.5 kb. 3D7 is the wild-type control and shows a band of the expected size (7.3 kb).
    Figure Legend Snippet: Generation of PfMyoB-GFP and PfMyoA-GFP parasites. A, schematic representation of the GFP-tagging of PfMyoB by single crossover homologous recombination into the myoB locus. The primers for PCR ( arrows 1 and 2 ) and the Southern blot probe together with restriction sites are labeled. X = XbaI and H = HpaI. B, diagnostic PCR on genomic DNA showing integration of PfMyoB-GFP ( primers 3 + 5 ) and wild type ( primers 3 + 4 ). Two PfMyoBGFP clones were examined. C, Southern blot analysis of cloned PfMyoB-GFP parasites. Genomic DNA was digested with XbaI and HpaI restriction enzymes. A probe to the myob region of homology showed the following: PfMyoB-GFP cycle 0 ( c0 ) shows the presence of wild-type (8.4 kb) and episome (4.3 kb) bands; 3D7 parasites only show the wild-type band. Clone 1 shows the expected bands for integration (7.9 and 4.8 kb), but also for episome, suggesting concatamer insertion. Clone 2 shows only bands for integration and was therefore used in all subsequent experiments. D, Western blot. Extracts of late stage schizonts from 3D7 and PfMyoB-GFP clone 2 parasites were immunoblotted wth an anti-GFP antibody. MyoB-GFP protein of ∼120 kDa was detected in clone 2. E, schematic representation of the GFP tagging of MyoA by single crossover homologous recombination into the myoA locus, with primers for PCR ( arrows with primer pair 15 and 16) and Southern blot probe and restriction sites labeled. C = ClaI and B = BsrFI. F, diagnostic PCR on genomic DNA showing integration of PfMyoA-GFP ( primers 17 + 5 ) and wild type ( primers 17 + 18 ). Four PfMyoA-GFP-expressing clones were examined. G, PfMyoA-GFP-expressing merozoites as viewed by live fluorescence microscopy. GFP was detected by green fluorescence, and the nuclei ( blue ) were labeled with Hoechst dye prior to microscopic analysis; the GFP signal is distributed to the parasite periphery. Scale bar, 2 μm. H, Southern blot analysis of cloned PfMyoA-GFP-expressing parasites. Genomic DNA was digested with ClaI and BsrFI. When probed with the myoa region of homology, all clones showed the expected two integration bands at 11.1 and 2.5 kb. 3D7 is the wild-type control and shows a band of the expected size (7.3 kb).

    Techniques Used: Homologous Recombination, Polymerase Chain Reaction, Southern Blot, Labeling, Diagnostic Assay, Clone Assay, Western Blot, Expressing, Fluorescence, Microscopy

    MyoB subcellular location in asexual blood and mosquito stages. A, microscopic analysis of live P. falciparum asexual blood stage parasites expressing GFP-tagged MyoB. Shown are blood stage parasites of increasing maturity from early schizogony (two to four nuclei; 30 h post-invasion) through to mature segmenter forms (44 h post-invasion) and free merozoites analyzed by fluorescence microscopy. GFP was detected by green fluorescence, and the nuclei ( blue ) were labeled with Hoechst dye prior to microscopic analysis. GFP fluorescence was not detected in trophozoites (data not shown) and early schizont stages, and was first apparent in mature multinucleate schizonts as a number of single dots (40 h post-invasion). Following cytokinesis, a single dot was present associated with each nucleus at what appeared to be the apical end of the cell. The images, merged with the differential interference contrast ( DIC ) image, are shown in the right panel. Scale bar, 2 μm. B, immunofluorescence of MyoB-HA in P. knowlesi schizonts. The epitope is detected using a specific antibody and an AlexaFluor 488 secondary antibody. Nuclei are labeled with DAPI; the green , blue, and differential interference contrast-merged images are also shown. Scale bar, 2 μm. C, expression of MyoB-GFP in the three invasive stages: schizonts (merozoites), ookinetes, and sporozoites of P. berghei . GFP is detected by green fluorescence, and nuclei ( blue ) were labeled with Hoechst dye. The merged and differential interference contrast images are also shown. The white arrows indicate expression of MyoB-GFP at the apical end of the parasites. Scale bar, 5 μm. D, expression of MyoB-GFP in liver stage schizonts of P. berghei , 55 h after invasion by a sporozoite. GFP was detected by green fluorescence, and the nuclei ( blue ) were labeled with Hoechst dye prior to microscopic analysis. Merged images are shown in the right panel. Scale bar, 10 μm. E, schematic showing the three invasive stages of Plasmodium , merozoite, ookinete, and sporozoite. The approximate length from anterior to posterior is shown.
    Figure Legend Snippet: MyoB subcellular location in asexual blood and mosquito stages. A, microscopic analysis of live P. falciparum asexual blood stage parasites expressing GFP-tagged MyoB. Shown are blood stage parasites of increasing maturity from early schizogony (two to four nuclei; 30 h post-invasion) through to mature segmenter forms (44 h post-invasion) and free merozoites analyzed by fluorescence microscopy. GFP was detected by green fluorescence, and the nuclei ( blue ) were labeled with Hoechst dye prior to microscopic analysis. GFP fluorescence was not detected in trophozoites (data not shown) and early schizont stages, and was first apparent in mature multinucleate schizonts as a number of single dots (40 h post-invasion). Following cytokinesis, a single dot was present associated with each nucleus at what appeared to be the apical end of the cell. The images, merged with the differential interference contrast ( DIC ) image, are shown in the right panel. Scale bar, 2 μm. B, immunofluorescence of MyoB-HA in P. knowlesi schizonts. The epitope is detected using a specific antibody and an AlexaFluor 488 secondary antibody. Nuclei are labeled with DAPI; the green , blue, and differential interference contrast-merged images are also shown. Scale bar, 2 μm. C, expression of MyoB-GFP in the three invasive stages: schizonts (merozoites), ookinetes, and sporozoites of P. berghei . GFP is detected by green fluorescence, and nuclei ( blue ) were labeled with Hoechst dye. The merged and differential interference contrast images are also shown. The white arrows indicate expression of MyoB-GFP at the apical end of the parasites. Scale bar, 5 μm. D, expression of MyoB-GFP in liver stage schizonts of P. berghei , 55 h after invasion by a sporozoite. GFP was detected by green fluorescence, and the nuclei ( blue ) were labeled with Hoechst dye prior to microscopic analysis. Merged images are shown in the right panel. Scale bar, 10 μm. E, schematic showing the three invasive stages of Plasmodium , merozoite, ookinete, and sporozoite. The approximate length from anterior to posterior is shown.

    Techniques Used: Expressing, Fluorescence, Microscopy, Labeling, Immunofluorescence

    PfMLC-B colocalizes in the cell with MyoB, binds to MyoB in vivo, and its C-terminal domain binds to the MyoB C-terminal sequence in vitro . A, structural model of amino acids 508–645 of PfMLC-B. The protein backbone is shown as a green ribbon , with a space-fill model of the structure overlaid. B, panel i, MLC-B-GFP subcellular location in a P. falciparum schizont. GFP was detected by green fluorescence, and the nuclei ( blue ) were labeled with Hoechst dye prior to microscopic analysis. Merged images are also shown. Scale bar, 2 μm. Panel ii, MLC-HA localization to the apex of merozoites in a P. knowlesi schizont. The HA epitope was detected using a specific antibody, followed by a species-specific AlexaFluor 488-labeled secondary antibody. Nuclei ( blue ) were detected using DAPI. Merged images are also shown. Scale bar, 2 μm. Panel iii, indirect immunofluorescence of P. falciparum schizont to determine colocalization of MLC-B-GFP ( green ) with MyoB ( red ). Nuclei were counterstained with DAPI ( blue ). The merged images are also shown. Scale bar, 2 μm. C, analysis of proteins affinity-purified with GFP-Trap from lysates of parasites expressing either PfMyoB-GFP or PfMLC-B-GFP by SDS-PAGE, trypsin digestion, and LC-MS/MS. Following subtraction of the list of proteins detected in control experiments, the lists of proteins detected in the two preparations were compared. Two proteins, MyoB and MLC-B, were in common. D, analysis of the binding of the C-terminal domain of PfMLC-B to peptides derived from the sequence at the C terminus of MyoB by either circular dichroism ( panels i and ii ) or thermal unfolding in the presence of peptides based on the MyoB amino acid sequences, residues Ile 763 to Arg 775 and Asn 780 to His 800 ( panel iii ). E, alignment of the neck regions of PfMyoA, PfMyoB, and TgMyoA showing confirmed ( red ) and speculated ( blue ) light-chain binding regions.
    Figure Legend Snippet: PfMLC-B colocalizes in the cell with MyoB, binds to MyoB in vivo, and its C-terminal domain binds to the MyoB C-terminal sequence in vitro . A, structural model of amino acids 508–645 of PfMLC-B. The protein backbone is shown as a green ribbon , with a space-fill model of the structure overlaid. B, panel i, MLC-B-GFP subcellular location in a P. falciparum schizont. GFP was detected by green fluorescence, and the nuclei ( blue ) were labeled with Hoechst dye prior to microscopic analysis. Merged images are also shown. Scale bar, 2 μm. Panel ii, MLC-HA localization to the apex of merozoites in a P. knowlesi schizont. The HA epitope was detected using a specific antibody, followed by a species-specific AlexaFluor 488-labeled secondary antibody. Nuclei ( blue ) were detected using DAPI. Merged images are also shown. Scale bar, 2 μm. Panel iii, indirect immunofluorescence of P. falciparum schizont to determine colocalization of MLC-B-GFP ( green ) with MyoB ( red ). Nuclei were counterstained with DAPI ( blue ). The merged images are also shown. Scale bar, 2 μm. C, analysis of proteins affinity-purified with GFP-Trap from lysates of parasites expressing either PfMyoB-GFP or PfMLC-B-GFP by SDS-PAGE, trypsin digestion, and LC-MS/MS. Following subtraction of the list of proteins detected in control experiments, the lists of proteins detected in the two preparations were compared. Two proteins, MyoB and MLC-B, were in common. D, analysis of the binding of the C-terminal domain of PfMLC-B to peptides derived from the sequence at the C terminus of MyoB by either circular dichroism ( panels i and ii ) or thermal unfolding in the presence of peptides based on the MyoB amino acid sequences, residues Ile 763 to Arg 775 and Asn 780 to His 800 ( panel iii ). E, alignment of the neck regions of PfMyoA, PfMyoB, and TgMyoA showing confirmed ( red ) and speculated ( blue ) light-chain binding regions.

    Techniques Used: In Vivo, Sequencing, In Vitro, Fluorescence, Labeling, Immunofluorescence, Affinity Purification, Expressing, SDS Page, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Binding Assay, Derivative Assay

    Generation of PkMyoB-HA and PbMyoB-GFP and detection of myob throughout the P. berghei life cycle. A, diagram representing C-terminal tagging of Pkmyob with a triple HA tag. Primers shown ( arrows ) were used to amplify the region of homology (6 and 7) for diagnostic PCR of WT parasite sequences (8 and 9) or parasites where integration had taken place (8 and 10). B, diagnostic PCR on genomic DNA showing integration of PkMyoB-HA into the myob locus in clone c1 with parental H.1 DNA as a control. C, Western blot with an anti-HA antibody on parental PkH.1 and PkMyoB-HA parasite lysates. In PkMyoB-HA parasites, a protein of ∼90 kDa is detected. The same blot was probed with an antibody against BiP to demonstrate equivalent sample loading. D, quantitative RT-PCR to show mRNA expression of Pbmyob during the parasite life cycle. Arginyl-tRNA synthetase and hsp70 were used as endogenous controls for normalization. Bars, three biological replicates, each ± S.E. AS, all asexual blood stages; Sch, schizonts; NG, nonactivated gametocytes; AG, activated gametocytes; Ook, ookinetes; Spz, sporozoites. E, diagram representing C-terminal tagging of Pb MyoB with GFP by single homologous recombination into endogenous myob gene, showing primers 11 and 12 to amplify the region of homology, and primers 13 and 14 to detect integration. F, confirmation of integration by PCR of Pbmyob-gfp using primer pair 13 and 14. G, Western blot of extracts of parasites expressing Pb MyoB-GFP or GFP.
    Figure Legend Snippet: Generation of PkMyoB-HA and PbMyoB-GFP and detection of myob throughout the P. berghei life cycle. A, diagram representing C-terminal tagging of Pkmyob with a triple HA tag. Primers shown ( arrows ) were used to amplify the region of homology (6 and 7) for diagnostic PCR of WT parasite sequences (8 and 9) or parasites where integration had taken place (8 and 10). B, diagnostic PCR on genomic DNA showing integration of PkMyoB-HA into the myob locus in clone c1 with parental H.1 DNA as a control. C, Western blot with an anti-HA antibody on parental PkH.1 and PkMyoB-HA parasite lysates. In PkMyoB-HA parasites, a protein of ∼90 kDa is detected. The same blot was probed with an antibody against BiP to demonstrate equivalent sample loading. D, quantitative RT-PCR to show mRNA expression of Pbmyob during the parasite life cycle. Arginyl-tRNA synthetase and hsp70 were used as endogenous controls for normalization. Bars, three biological replicates, each ± S.E. AS, all asexual blood stages; Sch, schizonts; NG, nonactivated gametocytes; AG, activated gametocytes; Ook, ookinetes; Spz, sporozoites. E, diagram representing C-terminal tagging of Pb MyoB with GFP by single homologous recombination into endogenous myob gene, showing primers 11 and 12 to amplify the region of homology, and primers 13 and 14 to detect integration. F, confirmation of integration by PCR of Pbmyob-gfp using primer pair 13 and 14. G, Western blot of extracts of parasites expressing Pb MyoB-GFP or GFP.

    Techniques Used: Diagnostic Assay, Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Expressing, Homologous Recombination

    PfMyoB-GFP does not associate with the glideosome components MTIP, GAP45, and GAP50. i, Western blot of parasite lysates from 3D7, MyoA-GFP ( A ), and MyoB-GFP ( B ) parasite lines. ii, GFP-TRAP immunoprecipitates from corresponding parasite lysates (shown in i ) separated by SDS-PAGE and probed with antibodies indicated on the right of each panel (rabbit anti-GFP, anti-GAP50, anti-GAP45, and anti-MTIP). Although GAP50, GAP45, and MTIP were present in all the lysates, they were detected in the MyoA-GFP immunoprecipitate but not in the MyoB-GFP immunoprecipitate. Molecular mass markers are indicated on the left in kDa.
    Figure Legend Snippet: PfMyoB-GFP does not associate with the glideosome components MTIP, GAP45, and GAP50. i, Western blot of parasite lysates from 3D7, MyoA-GFP ( A ), and MyoB-GFP ( B ) parasite lines. ii, GFP-TRAP immunoprecipitates from corresponding parasite lysates (shown in i ) separated by SDS-PAGE and probed with antibodies indicated on the right of each panel (rabbit anti-GFP, anti-GAP50, anti-GAP45, and anti-MTIP). Although GAP50, GAP45, and MTIP were present in all the lysates, they were detected in the MyoA-GFP immunoprecipitate but not in the MyoB-GFP immunoprecipitate. Molecular mass markers are indicated on the left in kDa.

    Techniques Used: Western Blot, SDS Page

    PfMyoB-GFP remains at the anterior of the merozoite during invasion of the host cell. MyoB-GFP-expressing P. falciparum parasites were fixed during various stages of invasion, and then MyoB-GFP ( green ) was revealed using rabbit anti-GFP antibodies, RON4 ( red ) was detected using mAb 24C6, and nuclei were stained with DAPI ( blue ). Merged images, including the differential interference contrast, are shown, as well as a schematic of the invasion stage in which the moving junction is shown by the blue arrowheads , the extracellular merozoite is gray , and the intracellular parasite is denoted by a dotted line and is uncolored. The invasion steps have been divided into initial attachment, followed by early and late stages of invasion as well as the final steps of invasion with the release of the remnant junction and formation of the ring stage. Scale bar, 2 μm.
    Figure Legend Snippet: PfMyoB-GFP remains at the anterior of the merozoite during invasion of the host cell. MyoB-GFP-expressing P. falciparum parasites were fixed during various stages of invasion, and then MyoB-GFP ( green ) was revealed using rabbit anti-GFP antibodies, RON4 ( red ) was detected using mAb 24C6, and nuclei were stained with DAPI ( blue ). Merged images, including the differential interference contrast, are shown, as well as a schematic of the invasion stage in which the moving junction is shown by the blue arrowheads , the extracellular merozoite is gray , and the intracellular parasite is denoted by a dotted line and is uncolored. The invasion steps have been divided into initial attachment, followed by early and late stages of invasion as well as the final steps of invasion with the release of the remnant junction and formation of the ring stage. Scale bar, 2 μm.

    Techniques Used: Expressing, Staining

    11) Product Images from "The Plasmodium Class XIV Myosin, MyoB, Has a Distinct Subcellular Location in Invasive and Motile Stages of the Malaria Parasite and an Unusual Light Chain *"

    Article Title: The Plasmodium Class XIV Myosin, MyoB, Has a Distinct Subcellular Location in Invasive and Motile Stages of the Malaria Parasite and an Unusual Light Chain *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.637694

    Generation of PfMyoB-GFP and PfMyoA-GFP parasites. A, schematic representation of the GFP-tagging of PfMyoB by single crossover homologous recombination into the myoB locus. The primers for PCR ( arrows 1 and 2 ) and the Southern blot probe together with restriction sites are labeled. X = XbaI and H = HpaI. B, diagnostic PCR on genomic DNA showing integration of PfMyoB-GFP ( primers 3 + 5 ) and wild type ( primers 3 + 4 ). Two PfMyoBGFP clones were examined. C, Southern blot analysis of cloned PfMyoB-GFP parasites. Genomic DNA was digested with XbaI and HpaI restriction enzymes. A probe to the myob region of homology showed the following: PfMyoB-GFP cycle 0 ( c0 ) shows the presence of wild-type (8.4 kb) and episome (4.3 kb) bands; 3D7 parasites only show the wild-type band. Clone 1 shows the expected bands for integration (7.9 and 4.8 kb), but also for episome, suggesting concatamer insertion. Clone 2 shows only bands for integration and was therefore used in all subsequent experiments. D, Western blot. Extracts of late stage schizonts from 3D7 and PfMyoB-GFP clone 2 parasites were immunoblotted wth an anti-GFP antibody. MyoB-GFP protein of ∼120 kDa was detected in clone 2. E, schematic representation of the GFP tagging of MyoA by single crossover homologous recombination into the myoA locus, with primers for PCR ( arrows with primer pair 15 and 16) and Southern blot probe and restriction sites labeled. C = ClaI and B = BsrFI. F, diagnostic PCR on genomic DNA showing integration of PfMyoA-GFP ( primers 17 + 5 ) and wild type ( primers 17 + 18 ). Four PfMyoA-GFP-expressing clones were examined. G, PfMyoA-GFP-expressing merozoites as viewed by live fluorescence microscopy. GFP was detected by green fluorescence, and the nuclei ( blue ) were labeled with Hoechst dye prior to microscopic analysis; the GFP signal is distributed to the parasite periphery. Scale bar, 2 μm. H, Southern blot analysis of cloned PfMyoA-GFP-expressing parasites. Genomic DNA was digested with ClaI and BsrFI. When probed with the myoa region of homology, all clones showed the expected two integration bands at 11.1 and 2.5 kb. 3D7 is the wild-type control and shows a band of the expected size (7.3 kb).
    Figure Legend Snippet: Generation of PfMyoB-GFP and PfMyoA-GFP parasites. A, schematic representation of the GFP-tagging of PfMyoB by single crossover homologous recombination into the myoB locus. The primers for PCR ( arrows 1 and 2 ) and the Southern blot probe together with restriction sites are labeled. X = XbaI and H = HpaI. B, diagnostic PCR on genomic DNA showing integration of PfMyoB-GFP ( primers 3 + 5 ) and wild type ( primers 3 + 4 ). Two PfMyoBGFP clones were examined. C, Southern blot analysis of cloned PfMyoB-GFP parasites. Genomic DNA was digested with XbaI and HpaI restriction enzymes. A probe to the myob region of homology showed the following: PfMyoB-GFP cycle 0 ( c0 ) shows the presence of wild-type (8.4 kb) and episome (4.3 kb) bands; 3D7 parasites only show the wild-type band. Clone 1 shows the expected bands for integration (7.9 and 4.8 kb), but also for episome, suggesting concatamer insertion. Clone 2 shows only bands for integration and was therefore used in all subsequent experiments. D, Western blot. Extracts of late stage schizonts from 3D7 and PfMyoB-GFP clone 2 parasites were immunoblotted wth an anti-GFP antibody. MyoB-GFP protein of ∼120 kDa was detected in clone 2. E, schematic representation of the GFP tagging of MyoA by single crossover homologous recombination into the myoA locus, with primers for PCR ( arrows with primer pair 15 and 16) and Southern blot probe and restriction sites labeled. C = ClaI and B = BsrFI. F, diagnostic PCR on genomic DNA showing integration of PfMyoA-GFP ( primers 17 + 5 ) and wild type ( primers 17 + 18 ). Four PfMyoA-GFP-expressing clones were examined. G, PfMyoA-GFP-expressing merozoites as viewed by live fluorescence microscopy. GFP was detected by green fluorescence, and the nuclei ( blue ) were labeled with Hoechst dye prior to microscopic analysis; the GFP signal is distributed to the parasite periphery. Scale bar, 2 μm. H, Southern blot analysis of cloned PfMyoA-GFP-expressing parasites. Genomic DNA was digested with ClaI and BsrFI. When probed with the myoa region of homology, all clones showed the expected two integration bands at 11.1 and 2.5 kb. 3D7 is the wild-type control and shows a band of the expected size (7.3 kb).

    Techniques Used: Homologous Recombination, Polymerase Chain Reaction, Southern Blot, Labeling, Diagnostic Assay, Clone Assay, Western Blot, Expressing, Fluorescence, Microscopy

    PfMyoB-GFP does not associate with the glideosome components MTIP, GAP45, and GAP50. i, Western blot of parasite lysates from 3D7, MyoA-GFP ( A ), and MyoB-GFP ( B ) parasite lines. ii, GFP-TRAP immunoprecipitates from corresponding parasite lysates (shown in i ) separated by SDS-PAGE and probed with antibodies indicated on the right of each panel (rabbit anti-GFP, anti-GAP50, anti-GAP45, and anti-MTIP). Although GAP50, GAP45, and MTIP were present in all the lysates, they were detected in the MyoA-GFP immunoprecipitate but not in the MyoB-GFP immunoprecipitate. Molecular mass markers are indicated on the left in kDa.
    Figure Legend Snippet: PfMyoB-GFP does not associate with the glideosome components MTIP, GAP45, and GAP50. i, Western blot of parasite lysates from 3D7, MyoA-GFP ( A ), and MyoB-GFP ( B ) parasite lines. ii, GFP-TRAP immunoprecipitates from corresponding parasite lysates (shown in i ) separated by SDS-PAGE and probed with antibodies indicated on the right of each panel (rabbit anti-GFP, anti-GAP50, anti-GAP45, and anti-MTIP). Although GAP50, GAP45, and MTIP were present in all the lysates, they were detected in the MyoA-GFP immunoprecipitate but not in the MyoB-GFP immunoprecipitate. Molecular mass markers are indicated on the left in kDa.

    Techniques Used: Western Blot, SDS Page

    12) Product Images from "CD83 Modulates B Cell Function In Vitro: Increased IL-10 and Reduced Ig Secretion by CD83Tg B Cells"

    Article Title: CD83 Modulates B Cell Function In Vitro: Increased IL-10 and Reduced Ig Secretion by CD83Tg B Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0000755

    Positive correlation of CD83 expression to CD86 and MHC-II expression on CD83Tg and CD83mu B cells. C57BL/6 and CD83 negative littermates to CD83Tg founder 1 mice (open bars), CD83Tg (black bars) or CD83mu (dark grey bars) derived spleen cells (2×10 6 /ml) were double stained for CD19 and either CD83 (2A), CD86 (2B), MHC-II (2C), CD80 (2D), CD40 (2E), CD69 (2F) or IgM (2G) ex vivo or after 48 h incubation with 10 µg/ml LPS as indicated on the x axis. 2×10 4 CD19 positive cells were analyzed by FACScan. Please note that each bar represents the combined results of five independent experiments employing two female age matched mice of each group per experiment, error bars show SEM. Asterisks indicate a significant difference of the mean (* p
    Figure Legend Snippet: Positive correlation of CD83 expression to CD86 and MHC-II expression on CD83Tg and CD83mu B cells. C57BL/6 and CD83 negative littermates to CD83Tg founder 1 mice (open bars), CD83Tg (black bars) or CD83mu (dark grey bars) derived spleen cells (2×10 6 /ml) were double stained for CD19 and either CD83 (2A), CD86 (2B), MHC-II (2C), CD80 (2D), CD40 (2E), CD69 (2F) or IgM (2G) ex vivo or after 48 h incubation with 10 µg/ml LPS as indicated on the x axis. 2×10 4 CD19 positive cells were analyzed by FACScan. Please note that each bar represents the combined results of five independent experiments employing two female age matched mice of each group per experiment, error bars show SEM. Asterisks indicate a significant difference of the mean (* p

    Techniques Used: Expressing, Mouse Assay, Derivative Assay, Staining, Ex Vivo, Incubation

    CD83 is upregulated on activated B cells. C57BL/6 mice derived spleen cells (2×10 6 /ml) were stimulated with anti-BCR (1 µg/ml) and IL-4 (20 ng/ml) or with LPS (10 µg/ml) as indicated in the headline. Cells were triple stained for CD19, CD83 and CD69 at the indicated time points. 1A: Dot blots show all lymphocytes positive for surface expression of CD83 on the x-axis and CD19 expression on the y-axis. 1BC: Graphs show the percentage of CD83 positive B cells (1B) or the mean fluorescence intensity (MFI) of CD83 on B cells (1C) after stimulation with anti-BCR alone (open circle) anti-BCR and IL-4 (closed circle) or with LPS (closed square) in an independent experiment, error bars show SEM of duplicates. 1D: Dot blot shows 2×10 4 CD19 positive cells derived from LPS activated spleen cells analyzed for CD83 (x-axis) and CD69 (y-axis) surface expression. 1E: 2×10 6 purified C57BL/6 spleen derived B cells were stimulated with LPS (10 µg/ml). B cells were lysed at the indicated time points, deglycosylated and separated by SDS-PAGE. CD83 was detected by western blot with a polyclonal rabbit anti-CD83 serum. Results are representative for at least three independent experiments.
    Figure Legend Snippet: CD83 is upregulated on activated B cells. C57BL/6 mice derived spleen cells (2×10 6 /ml) were stimulated with anti-BCR (1 µg/ml) and IL-4 (20 ng/ml) or with LPS (10 µg/ml) as indicated in the headline. Cells were triple stained for CD19, CD83 and CD69 at the indicated time points. 1A: Dot blots show all lymphocytes positive for surface expression of CD83 on the x-axis and CD19 expression on the y-axis. 1BC: Graphs show the percentage of CD83 positive B cells (1B) or the mean fluorescence intensity (MFI) of CD83 on B cells (1C) after stimulation with anti-BCR alone (open circle) anti-BCR and IL-4 (closed circle) or with LPS (closed square) in an independent experiment, error bars show SEM of duplicates. 1D: Dot blot shows 2×10 4 CD19 positive cells derived from LPS activated spleen cells analyzed for CD83 (x-axis) and CD69 (y-axis) surface expression. 1E: 2×10 6 purified C57BL/6 spleen derived B cells were stimulated with LPS (10 µg/ml). B cells were lysed at the indicated time points, deglycosylated and separated by SDS-PAGE. CD83 was detected by western blot with a polyclonal rabbit anti-CD83 serum. Results are representative for at least three independent experiments.

    Techniques Used: Mouse Assay, Derivative Assay, Staining, Expressing, Fluorescence, Dot Blot, Purification, SDS Page, Western Blot

    13) Product Images from "Type I interferon signaling is required for activation of the inflammasome during Francisella infection"

    Article Title: Type I interferon signaling is required for activation of the inflammasome during Francisella infection

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20062665

    Type I IFN signaling is necessary for activation of the inflammasome during F. novicida and L. monocytogenes infections but not upon ATP treatment. (A) WT, IFNR −/− , and ASC −/− BMMs uninfected (Un) or infected with mglA (m) or WT F. novicida (W) were lysed at 9 h PI. Caspase-1 processing was visualized by detection of the p20 subunit only in WT macrophages infected with WT F. novicida . (B and C) IL-1β (B) and -18 (C) were quantified by ELISA in the supernatant of preactivated BMMs. Similar levels were detected in WT and IFNR −/− BMMs treated for 3 h with 5 mM ATP (left), whereas high levels were only detected in WT macrophages upon infection with F. novicida for 5 h (right). (D) Cell death (left) was strongly reduced in IFNR −/− compared with WT BMMs at 6 h PI with L. monocytogenes . Similarly, IL-1β (middle) and -18 (right) levels were lower in activated BMMs infected for 2.5 h with L. monocytogenes at the indicated MOI.
    Figure Legend Snippet: Type I IFN signaling is necessary for activation of the inflammasome during F. novicida and L. monocytogenes infections but not upon ATP treatment. (A) WT, IFNR −/− , and ASC −/− BMMs uninfected (Un) or infected with mglA (m) or WT F. novicida (W) were lysed at 9 h PI. Caspase-1 processing was visualized by detection of the p20 subunit only in WT macrophages infected with WT F. novicida . (B and C) IL-1β (B) and -18 (C) were quantified by ELISA in the supernatant of preactivated BMMs. Similar levels were detected in WT and IFNR −/− BMMs treated for 3 h with 5 mM ATP (left), whereas high levels were only detected in WT macrophages upon infection with F. novicida for 5 h (right). (D) Cell death (left) was strongly reduced in IFNR −/− compared with WT BMMs at 6 h PI with L. monocytogenes . Similarly, IL-1β (middle) and -18 (right) levels were lower in activated BMMs infected for 2.5 h with L. monocytogenes at the indicated MOI.

    Techniques Used: Activation Assay, Infection, Enzyme-linked Immunosorbent Assay

    Type I IFN induction and signaling is required for F. novicida –mediated but not for S. typhimurium –mediated cell death. Cell death of WT, IFNR −/− , ASC −/− , and caspase-1 (casp1) −/− BMMs was assayed by lactate dehydrogenase (LDH) release. BMMs either unactivated (B) or preactivated with heat-killed F. novicida (A, C, and D [left]) or pretreated with recombinant IFN-β (D, right) were infected for 8 (A), 12.5 (B), 3 (C), or 6 h (D) with F. novicida (A, B, and D) or S. typhimurium (C) strains at the indicated MOI. In agreement with previous data ( 30 ), cell death required the S. typhimurium gene sipB . Error bars represent SEM.
    Figure Legend Snippet: Type I IFN induction and signaling is required for F. novicida –mediated but not for S. typhimurium –mediated cell death. Cell death of WT, IFNR −/− , ASC −/− , and caspase-1 (casp1) −/− BMMs was assayed by lactate dehydrogenase (LDH) release. BMMs either unactivated (B) or preactivated with heat-killed F. novicida (A, C, and D [left]) or pretreated with recombinant IFN-β (D, right) were infected for 8 (A), 12.5 (B), 3 (C), or 6 h (D) with F. novicida (A, B, and D) or S. typhimurium (C) strains at the indicated MOI. In agreement with previous data ( 30 ), cell death required the S. typhimurium gene sipB . Error bars represent SEM.

    Techniques Used: Recombinant, Infection

    F. novicida in the host cytosol induces IFN-β secretion in a TLR-independent IRF-3–dependent manner. IFN-β mRNA levels were determined by quantitative RT-PCR in uninfected BMMs (Un) or at various times PI (A) and 7 (C), 9 (D), or 8 (E) h PI with either mglA or WT F. novicida . IFN-β levels were determined by ELISA in the supernatant of BMMs infected at the indicated MOI for 9 h (ND, nondetectable; B). Various vacuole-restricted mutants do not induce IFN-β mRNA, whereas their complemented counterparts (c.) do. Cytosolic localization is shown (C). BMMs from WT or from IRF-3 −/− , ASC −/− , MyD88/TRIF DKO , Ipaf −/− , RIP2 −/− (D), MAVS −/− mice, or WT littermates (E) were analyzed for their IFN-β mRNA levels. Error bars represent SEM.
    Figure Legend Snippet: F. novicida in the host cytosol induces IFN-β secretion in a TLR-independent IRF-3–dependent manner. IFN-β mRNA levels were determined by quantitative RT-PCR in uninfected BMMs (Un) or at various times PI (A) and 7 (C), 9 (D), or 8 (E) h PI with either mglA or WT F. novicida . IFN-β levels were determined by ELISA in the supernatant of BMMs infected at the indicated MOI for 9 h (ND, nondetectable; B). Various vacuole-restricted mutants do not induce IFN-β mRNA, whereas their complemented counterparts (c.) do. Cytosolic localization is shown (C). BMMs from WT or from IRF-3 −/− , ASC −/− , MyD88/TRIF DKO , Ipaf −/− , RIP2 −/− (D), MAVS −/− mice, or WT littermates (E) were analyzed for their IFN-β mRNA levels. Error bars represent SEM.

    Techniques Used: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Infection, Mouse Assay

    14) Product Images from "Extracellular vesicles in DLBCL provide abundant clues to aberrant transcriptional programming and genomic alterations"

    Article Title: Extracellular vesicles in DLBCL provide abundant clues to aberrant transcriptional programming and genomic alterations

    Journal: Blood

    doi: 10.1182/blood-2017-12-821843

    Isolation of EVs from DLBCL cell lines and primary sample. (A) Size distribution and concentration of EVs isolated from OCI-Ly1 cell line (top) and a primary DLBCL sample of GCB subtype (bottom) by ultracentrifugation determined by the NanoSight instrument. (B) Western blot for the exosomal proteins ALIX (100 kDa), TSG101 (46 kDa), CD81 (26 kDa), and as a negative control, HSP90B1 protein (90 kDa). (C) FACS analysis of exosomal surface markers demonstrates expression of CD63 in OCI-Ly1–derived EVs but not in the parental whole cells, expression of B-cell surface marker CD20, GC marker CD10, but not T-cell marker CD4 in both derived EVs and parental OCI-Ly1 cells. (D) Confocal fluorescence microscope image shows uptake of EVs released by lymphoma OCI-LY1 cells (green) by stromal HK cells (blue = DAPI; red = α-actin).
    Figure Legend Snippet: Isolation of EVs from DLBCL cell lines and primary sample. (A) Size distribution and concentration of EVs isolated from OCI-Ly1 cell line (top) and a primary DLBCL sample of GCB subtype (bottom) by ultracentrifugation determined by the NanoSight instrument. (B) Western blot for the exosomal proteins ALIX (100 kDa), TSG101 (46 kDa), CD81 (26 kDa), and as a negative control, HSP90B1 protein (90 kDa). (C) FACS analysis of exosomal surface markers demonstrates expression of CD63 in OCI-Ly1–derived EVs but not in the parental whole cells, expression of B-cell surface marker CD20, GC marker CD10, but not T-cell marker CD4 in both derived EVs and parental OCI-Ly1 cells. (D) Confocal fluorescence microscope image shows uptake of EVs released by lymphoma OCI-LY1 cells (green) by stromal HK cells (blue = DAPI; red = α-actin).

    Techniques Used: Isolation, Concentration Assay, Western Blot, Negative Control, FACS, Expressing, Derivative Assay, Marker, Fluorescence, Microscopy

    RNA species found in EVs and cells. (A) DLBCL cell lines OCI-Ly3, HBL1, TMD8, OCI-Ly1, and OCI-Ly7, (B) EVs from DLBCL cell lines OCI-Ly3, HBL1, TMD8, OCI-Ly1, and OCI-Ly7, (C) whole cell DLBCL primary samples DLBCL1, DLBCL2, DLBCL3, DLBCL4, DLBCL5, (D) EVs from DLBCL primary sample DLBCL6, (E) whole cell normal B cells NB1, NB2, and NB3, and (F) EVs from normal B cells B2. EVs contain a variety of RNA species with enrichment for noncoding RNAs, long intergenic noncoding RNAs (lincRNAs), snoRNAs, and snRNAs. Quantities are displayed as the sum of average transcripts per million (TPM) per RNA class. rRNA, ribosomal RNA.
    Figure Legend Snippet: RNA species found in EVs and cells. (A) DLBCL cell lines OCI-Ly3, HBL1, TMD8, OCI-Ly1, and OCI-Ly7, (B) EVs from DLBCL cell lines OCI-Ly3, HBL1, TMD8, OCI-Ly1, and OCI-Ly7, (C) whole cell DLBCL primary samples DLBCL1, DLBCL2, DLBCL3, DLBCL4, DLBCL5, (D) EVs from DLBCL primary sample DLBCL6, (E) whole cell normal B cells NB1, NB2, and NB3, and (F) EVs from normal B cells B2. EVs contain a variety of RNA species with enrichment for noncoding RNAs, long intergenic noncoding RNAs (lincRNAs), snoRNAs, and snRNAs. Quantities are displayed as the sum of average transcripts per million (TPM) per RNA class. rRNA, ribosomal RNA.

    Techniques Used:

    EV exchange by DLBCL cell lines and uptake by normal tonsillar cells. (A) Positive SYTO RNA fluorescent stain of supernatant from recipient OCI-Ly3 cells previously incubated with EVs from stained donor OCI-Ly1 cells (left panel) and positive stain for the general exosome marker CD63 (right panel), indicating uptake of RNAs from stained OCI-Ly1 donor cells. (B) Control negative SYTO RNA fluorescent staining of supernatant from recipient OCI-Ly3 cells incubated with EVs of unstained OCI-Ly1 donor cells (left panel), and positive staining for CD63 (right panel). (C) Positive SYTO RNA fluorescent stain in recipient normal tonsillar lymphocytes incubated with EVs isolated from previously stained donor OCI-Ly1 (left panel) or HBL1 (right panel).
    Figure Legend Snippet: EV exchange by DLBCL cell lines and uptake by normal tonsillar cells. (A) Positive SYTO RNA fluorescent stain of supernatant from recipient OCI-Ly3 cells previously incubated with EVs from stained donor OCI-Ly1 cells (left panel) and positive stain for the general exosome marker CD63 (right panel), indicating uptake of RNAs from stained OCI-Ly1 donor cells. (B) Control negative SYTO RNA fluorescent staining of supernatant from recipient OCI-Ly3 cells incubated with EVs of unstained OCI-Ly1 donor cells (left panel), and positive staining for CD63 (right panel). (C) Positive SYTO RNA fluorescent stain in recipient normal tonsillar lymphocytes incubated with EVs isolated from previously stained donor OCI-Ly1 (left panel) or HBL1 (right panel).

    Techniques Used: Staining, Incubation, Marker, Isolation

    15) Product Images from "Molecular chaperones and the assembly of the prion Sup35p, an in vitro study"

    Article Title: Molecular chaperones and the assembly of the prion Sup35p, an in vitro study

    Journal: The EMBO Journal

    doi: 10.1038/sj.emboj.7600985

    Functional interplay of molecular chaperones during the assembly of Sup35p. (A–D) Time courses of Sup35p (4 μM) assembly at 10°C in the absence (•) and ( A ) the presence of equimolar amounts of Ydj1p/Ssa1p (▴) and Sis1p/Ssa1p (□), ( B ) Ydj1p/Hsp104p (▴) and Sis1p/Hsp104p (♦), ( C ) Ssa1p/Hsp104p (▴) and ( D ) Ydj1p/Ssa1p/Hsp104p (▴), Sis1p/Ssa1p/Hsp104p (♦). Assembly was monitored by thioflavin T binding. AU, arbitrary units.
    Figure Legend Snippet: Functional interplay of molecular chaperones during the assembly of Sup35p. (A–D) Time courses of Sup35p (4 μM) assembly at 10°C in the absence (•) and ( A ) the presence of equimolar amounts of Ydj1p/Ssa1p (▴) and Sis1p/Ssa1p (□), ( B ) Ydj1p/Hsp104p (▴) and Sis1p/Hsp104p (♦), ( C ) Ssa1p/Hsp104p (▴) and ( D ) Ydj1p/Ssa1p/Hsp104p (▴), Sis1p/Ssa1p/Hsp104p (♦). Assembly was monitored by thioflavin T binding. AU, arbitrary units.

    Techniques Used: Functional Assay, Binding Assay

    Gel filtration analysis of the interaction of molecular chaperones with soluble Sup35p. (A–J) Analysis on 10% SDS–PAGE of protein fractions emerging from a Superose 12 HR column equilibrated and eluted by assembly buffer, (A–I) containing 2 mM ATP, (J) without ATP. ( A ) Sup35p; ( B ) Ssa1p; ( C ) Ydj1p; ( D ) Sis1p; ( E ) Sup35p and Ssa1p; ( F ) Sup35p and Ydj1p; ( G ) Sup35p and Sis1p; ( H ) Sup35p, Ssa1p and Ydj1p; and ( I , J ) Sup35p, Ssa1p and Sis1p. All proteins were loaded at 4 μM. Arrows show the location of molecular size markers (aldolase, 158 kDa; bovine albumin, 66 kDa; ovalbumin, 43 kDa and carboxy anhydrase, 29.5 kDa) run under identical conditions on the same column. V o (blue dextran, 2000 kDa) is the void volume of the column.
    Figure Legend Snippet: Gel filtration analysis of the interaction of molecular chaperones with soluble Sup35p. (A–J) Analysis on 10% SDS–PAGE of protein fractions emerging from a Superose 12 HR column equilibrated and eluted by assembly buffer, (A–I) containing 2 mM ATP, (J) without ATP. ( A ) Sup35p; ( B ) Ssa1p; ( C ) Ydj1p; ( D ) Sis1p; ( E ) Sup35p and Ssa1p; ( F ) Sup35p and Ydj1p; ( G ) Sup35p and Sis1p; ( H ) Sup35p, Ssa1p and Ydj1p; and ( I , J ) Sup35p, Ssa1p and Sis1p. All proteins were loaded at 4 μM. Arrows show the location of molecular size markers (aldolase, 158 kDa; bovine albumin, 66 kDa; ovalbumin, 43 kDa and carboxy anhydrase, 29.5 kDa) run under identical conditions on the same column. V o (blue dextran, 2000 kDa) is the void volume of the column.

    Techniques Used: Filtration, SDS Page

    Assembly of Sup35p in the presence of individual molecular chaperones. ( A , B ) Time courses of Sup35p (4 μM) assembly at 10°C in the absence of molecular chaperones (•) and in the presence of submolar, 0.4 μM (open symbols), and equimolar (filled symbols) concentrations of Ssa1p (□, ▪), Sis1p (▵, ▴) and Hsp82p (⋄, ♦). ( C ) Time courses of Sup35p (4 μM) assembly in the absence (•) and in the presence of submolar 0.4 μM (▾, ♦) and equimolar (▴, ▪) concentrations of Ydj1p and Hsp104p, respectively. All assembly reactions were monitored by thioflavin T binding. AU, arbitrary units.
    Figure Legend Snippet: Assembly of Sup35p in the presence of individual molecular chaperones. ( A , B ) Time courses of Sup35p (4 μM) assembly at 10°C in the absence of molecular chaperones (•) and in the presence of submolar, 0.4 μM (open symbols), and equimolar (filled symbols) concentrations of Ssa1p (□, ▪), Sis1p (▵, ▴) and Hsp82p (⋄, ♦). ( C ) Time courses of Sup35p (4 μM) assembly in the absence (•) and in the presence of submolar 0.4 μM (▾, ♦) and equimolar (▴, ▪) concentrations of Ydj1p and Hsp104p, respectively. All assembly reactions were monitored by thioflavin T binding. AU, arbitrary units.

    Techniques Used: Binding Assay

    16) Product Images from "PDGF-B-driven gliomagenesis can occur in the absence of the proteoglycan NG2"

    Article Title: PDGF-B-driven gliomagenesis can occur in the absence of the proteoglycan NG2

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-10-550

    Cells derived from PDGF-B-induced gliomas are tumorigenic also in the absence of NG2 expression . (A) Survival curves following transplantation into adult brains of PDGF-B-induced tumor cells derived from wild type (black line) or NG2-KO (green line) mice. The Log-rank test performed on the two survival distributions showed that there are no significant differences (Log-rank Test = N.S.). (B) Fluorescence image of a NG2-KO secondary tumor. (C-F) Coronal sections of secondary tumors derived from NG2-KO (C-D) or wild type (E-F) mice stained with DAPI (C, E) or hematoxylin and eosin (D, F). NT: non-tumor zone; T: highly cellularized tumor zone; Dashed contours highlight the tumor mass; arrows point pseudopalisade structures. Scale bars: 1 mm (B); 100 μm (C-F).
    Figure Legend Snippet: Cells derived from PDGF-B-induced gliomas are tumorigenic also in the absence of NG2 expression . (A) Survival curves following transplantation into adult brains of PDGF-B-induced tumor cells derived from wild type (black line) or NG2-KO (green line) mice. The Log-rank test performed on the two survival distributions showed that there are no significant differences (Log-rank Test = N.S.). (B) Fluorescence image of a NG2-KO secondary tumor. (C-F) Coronal sections of secondary tumors derived from NG2-KO (C-D) or wild type (E-F) mice stained with DAPI (C, E) or hematoxylin and eosin (D, F). NT: non-tumor zone; T: highly cellularized tumor zone; Dashed contours highlight the tumor mass; arrows point pseudopalisade structures. Scale bars: 1 mm (B); 100 μm (C-F).

    Techniques Used: Derivative Assay, Expressing, Transplantation Assay, Mouse Assay, Fluorescence, Staining

    NG2 silencing does not impair the tumorigenic potential of cells derived from PDGF-B-induced tumors . (A-B) Immunofluorescence stainings of PDGF-B-induced glioma cultures transduced with miR-NG2 (A) or miRneg (B) and stained with anti-GFP antibody in green, anti-NG2 antibody in red and DAPI (nuclei) in blue. (C-D) Histograms showing the efficiency of miR-NG2 silencing in PDGF-B-induced glioma cultures (C) or in cells extracted from a secondary tumor generated by the intracranial injection of PDGF-B-induced glioma cells previously transduced with miR-NG2 (miR-NG2 secondary tumor; D). GFP positive cells represent glioma cells which were actually transduced with miR-NG2, while GFP-negative cells were not transduced. (E) Fluorescence image of a miR-NG2 secondary tumor. (F) Immunofluorescence staining of miR-NG2 secondary tumor cells with anti-GFP antibody in green, anti-NG2 antibody in red and DAPI (nuclei) in blue. Scale bars: 50 μm (A-B, F); 0.5 mm (E).
    Figure Legend Snippet: NG2 silencing does not impair the tumorigenic potential of cells derived from PDGF-B-induced tumors . (A-B) Immunofluorescence stainings of PDGF-B-induced glioma cultures transduced with miR-NG2 (A) or miRneg (B) and stained with anti-GFP antibody in green, anti-NG2 antibody in red and DAPI (nuclei) in blue. (C-D) Histograms showing the efficiency of miR-NG2 silencing in PDGF-B-induced glioma cultures (C) or in cells extracted from a secondary tumor generated by the intracranial injection of PDGF-B-induced glioma cells previously transduced with miR-NG2 (miR-NG2 secondary tumor; D). GFP positive cells represent glioma cells which were actually transduced with miR-NG2, while GFP-negative cells were not transduced. (E) Fluorescence image of a miR-NG2 secondary tumor. (F) Immunofluorescence staining of miR-NG2 secondary tumor cells with anti-GFP antibody in green, anti-NG2 antibody in red and DAPI (nuclei) in blue. Scale bars: 50 μm (A-B, F); 0.5 mm (E).

    Techniques Used: Derivative Assay, Immunofluorescence, Transduction, Staining, Generated, Injection, Fluorescence

    PDGF-B overexpression induced gliomas in NG2-KO mice . (A) Survival curves following PDGF-B embryonic transduction of NG2-KO (green line) and wild type (black line) mice. The Log-rank test performed on the survival curves of NG2-KO and wild type animals showed that there are no significant differences (Log-rank Test = N.S.) between the two distributions. (B-C) Fluorescence images of PDGF-B-induced tumors in NG2-KO (B) and wild type (C) mice. (D-M) Coronal sections of tumors obtained in NG2-KO (D, F, F', H, I) or wild type (E, G, G', L, M) mice stained with DAPI for nuclear staining in blue and antibodies for the indicated antigens (D-G', I, M) or with hematoxylin and eosin (H, L). F' and G' are magnifications of the insets in figures F and G respectively, arrowheads point infiltrating cells. NT: non-tumor zone; T: highly cellularized tumor zone. Scale bars: 0.5 mm (B-E); 100 μm (F-M).
    Figure Legend Snippet: PDGF-B overexpression induced gliomas in NG2-KO mice . (A) Survival curves following PDGF-B embryonic transduction of NG2-KO (green line) and wild type (black line) mice. The Log-rank test performed on the survival curves of NG2-KO and wild type animals showed that there are no significant differences (Log-rank Test = N.S.) between the two distributions. (B-C) Fluorescence images of PDGF-B-induced tumors in NG2-KO (B) and wild type (C) mice. (D-M) Coronal sections of tumors obtained in NG2-KO (D, F, F', H, I) or wild type (E, G, G', L, M) mice stained with DAPI for nuclear staining in blue and antibodies for the indicated antigens (D-G', I, M) or with hematoxylin and eosin (H, L). F' and G' are magnifications of the insets in figures F and G respectively, arrowheads point infiltrating cells. NT: non-tumor zone; T: highly cellularized tumor zone. Scale bars: 0.5 mm (B-E); 100 μm (F-M).

    Techniques Used: Over Expression, Mouse Assay, Transduction, Fluorescence, Staining

    PDGF-B overexpression induced oligodendrogliomas in NG2-KO mice . (A-H) Immunofluorescence stainings of NG2-KO (A, C, E, G) and wild type (B, D, F, H) glioma sections with anti-GFP antibody in green, DAPI for nuclear staining in blue and antibodies for the indicated antigens in red. Scale bar: 50 μm
    Figure Legend Snippet: PDGF-B overexpression induced oligodendrogliomas in NG2-KO mice . (A-H) Immunofluorescence stainings of NG2-KO (A, C, E, G) and wild type (B, D, F, H) glioma sections with anti-GFP antibody in green, DAPI for nuclear staining in blue and antibodies for the indicated antigens in red. Scale bar: 50 μm

    Techniques Used: Over Expression, Mouse Assay, Immunofluorescence, Staining

    PDGF-B-induced tumors recruit OPCs also in the absence of NG2 expression . (A-H) Immunofluorescence stainings of NG2-KO (A-D) and wild type (E-H) glioma sections with anti-GFP antibody in green, DAPI for nuclear staining in blue and antibodies for the indicated antigens in red. The left column of the figure corresponds to tumor infiltrated regions, while pictures on the right represent normal brain regions next to tumor areas showing the basal level of OPC markers expression for comparison. Scale bars: 50 μm
    Figure Legend Snippet: PDGF-B-induced tumors recruit OPCs also in the absence of NG2 expression . (A-H) Immunofluorescence stainings of NG2-KO (A-D) and wild type (E-H) glioma sections with anti-GFP antibody in green, DAPI for nuclear staining in blue and antibodies for the indicated antigens in red. The left column of the figure corresponds to tumor infiltrated regions, while pictures on the right represent normal brain regions next to tumor areas showing the basal level of OPC markers expression for comparison. Scale bars: 50 μm

    Techniques Used: Expressing, Immunofluorescence, Staining

    PDGF-B-induced tumors can be propagated in culture also in the absence of NG2 expression .(A-H) Immunofluorescence stainings of NG2-KO (A-D) and wild type (E-H) glioma cultures with anti-GFP antibody in green, DAPI for nuclear staining in blue and the antibody for the indicated antigen in red. (I) Histogram showing the percentage of cells expressing the indicated markers in NG2-KO (red bars) and wild type (blue bars) glioma cell cultures. (L-M) Bright field microphotographs of NG2-KO (L) and wild type (M) glioma cells showing the ability to form foci in vitro. Scale bars: 50 μm (A-D); 100 μm (L-M).
    Figure Legend Snippet: PDGF-B-induced tumors can be propagated in culture also in the absence of NG2 expression .(A-H) Immunofluorescence stainings of NG2-KO (A-D) and wild type (E-H) glioma cultures with anti-GFP antibody in green, DAPI for nuclear staining in blue and the antibody for the indicated antigen in red. (I) Histogram showing the percentage of cells expressing the indicated markers in NG2-KO (red bars) and wild type (blue bars) glioma cell cultures. (L-M) Bright field microphotographs of NG2-KO (L) and wild type (M) glioma cells showing the ability to form foci in vitro. Scale bars: 50 μm (A-D); 100 μm (L-M).

    Techniques Used: Expressing, Immunofluorescence, Staining, In Vitro

    PDGF-B transduction induces an oligodendroglial fate in cultured neural progenitor cells . (A-B, left side) Histograms showing the percentage of Olig2-positive cells in PDGF-B (red bars) and control (green bars) transduced cortical (A) and GE (B) cultures obtained from wild type and NG2-KO embryos. (A-B, right side) Immunofluorescence micrographs of cortical (A) and GE (B) cultures derived from wild type and NG2-KO mice following the transduction with PDGF-B/GFP or control retroviruses. The immunoreactivity to Olig2 and GFP is shown in red and in green, respectively. NG2-KO cultures: nExp = 3; Wild type cultures: nExp = 2. *p
    Figure Legend Snippet: PDGF-B transduction induces an oligodendroglial fate in cultured neural progenitor cells . (A-B, left side) Histograms showing the percentage of Olig2-positive cells in PDGF-B (red bars) and control (green bars) transduced cortical (A) and GE (B) cultures obtained from wild type and NG2-KO embryos. (A-B, right side) Immunofluorescence micrographs of cortical (A) and GE (B) cultures derived from wild type and NG2-KO mice following the transduction with PDGF-B/GFP or control retroviruses. The immunoreactivity to Olig2 and GFP is shown in red and in green, respectively. NG2-KO cultures: nExp = 3; Wild type cultures: nExp = 2. *p

    Techniques Used: Transduction, Cell Culture, Immunofluorescence, Derivative Assay, Mouse Assay

    17) Product Images from "Molecular chaperones and the assembly of the prion Sup35p, an in vitro study"

    Article Title: Molecular chaperones and the assembly of the prion Sup35p, an in vitro study

    Journal: The EMBO Journal

    doi: 10.1038/sj.emboj.7600985

    Functional interplay of molecular chaperones during the assembly of Sup35p. (A–D) Time courses of Sup35p (4 μM) assembly at 10°C in the absence (•) and ( A ) the presence of equimolar amounts of Ydj1p/Ssa1p (▴) and Sis1p/Ssa1p (□), ( B ) Ydj1p/Hsp104p (▴) and Sis1p/Hsp104p (♦), ( C ) Ssa1p/Hsp104p (▴) and ( D ) Ydj1p/Ssa1p/Hsp104p (▴), Sis1p/Ssa1p/Hsp104p (♦). Assembly was monitored by thioflavin T binding. AU, arbitrary units.
    Figure Legend Snippet: Functional interplay of molecular chaperones during the assembly of Sup35p. (A–D) Time courses of Sup35p (4 μM) assembly at 10°C in the absence (•) and ( A ) the presence of equimolar amounts of Ydj1p/Ssa1p (▴) and Sis1p/Ssa1p (□), ( B ) Ydj1p/Hsp104p (▴) and Sis1p/Hsp104p (♦), ( C ) Ssa1p/Hsp104p (▴) and ( D ) Ydj1p/Ssa1p/Hsp104p (▴), Sis1p/Ssa1p/Hsp104p (♦). Assembly was monitored by thioflavin T binding. AU, arbitrary units.

    Techniques Used: Functional Assay, Binding Assay

    Schematic representation of the assembly of Sup35p into insoluble high molecular weight oligomers and the effect of the different molecular chaperones used on the assembly reaction. Soluble assembly-competent Sup35p form presumably through a reversible unfolding event, which extent is unknown. This assembly-competent form oligomerizes into soluble high molecular weight oligomeric forms that can either grow by incorporation of soluble assembly-competent Sup35p or by condensation yielding insoluble high molecular weight forms, for example, fibrils. Hsp104p promotes the formation of the soluble high molecular weight oligomers that act as nuclei in Sup35p assembly. Ydj1p binds either soluble Sup35p folding intermediates or assembly-competent Sup35p, thus forming a Sup35p–Ydj1p assembly-incompetent binary complex. Ssa1p on its own, in the presence or the absence of ATP, has no significant influence on the assembly pathway. It is only in the presence of its Hsp40 cochaperone and ATP that an assembly-incompetent ternary complex (Sup35p–Ssa1p–Ydj1p or Sis1p) forms. The width of each arrow depicts the strength with which the assembly pathway is displaced from its normal fate.
    Figure Legend Snippet: Schematic representation of the assembly of Sup35p into insoluble high molecular weight oligomers and the effect of the different molecular chaperones used on the assembly reaction. Soluble assembly-competent Sup35p form presumably through a reversible unfolding event, which extent is unknown. This assembly-competent form oligomerizes into soluble high molecular weight oligomeric forms that can either grow by incorporation of soluble assembly-competent Sup35p or by condensation yielding insoluble high molecular weight forms, for example, fibrils. Hsp104p promotes the formation of the soluble high molecular weight oligomers that act as nuclei in Sup35p assembly. Ydj1p binds either soluble Sup35p folding intermediates or assembly-competent Sup35p, thus forming a Sup35p–Ydj1p assembly-incompetent binary complex. Ssa1p on its own, in the presence or the absence of ATP, has no significant influence on the assembly pathway. It is only in the presence of its Hsp40 cochaperone and ATP that an assembly-incompetent ternary complex (Sup35p–Ssa1p–Ydj1p or Sis1p) forms. The width of each arrow depicts the strength with which the assembly pathway is displaced from its normal fate.

    Techniques Used: Molecular Weight, Activated Clotting Time Assay

    Assembly of Sup35p in the presence of individual molecular chaperones. ( A , B ) Time courses of Sup35p (4 μM) assembly at 10°C in the absence of molecular chaperones (•) and in the presence of submolar, 0.4 μM (open symbols), and equimolar (filled symbols) concentrations of Ssa1p (□, ▪), Sis1p (▵, ▴) and Hsp82p (⋄, ♦). ( C ) Time courses of Sup35p (4 μM) assembly in the absence (•) and in the presence of submolar 0.4 μM (▾, ♦) and equimolar (▴, ▪) concentrations of Ydj1p and Hsp104p, respectively. All assembly reactions were monitored by thioflavin T binding. AU, arbitrary units.
    Figure Legend Snippet: Assembly of Sup35p in the presence of individual molecular chaperones. ( A , B ) Time courses of Sup35p (4 μM) assembly at 10°C in the absence of molecular chaperones (•) and in the presence of submolar, 0.4 μM (open symbols), and equimolar (filled symbols) concentrations of Ssa1p (□, ▪), Sis1p (▵, ▴) and Hsp82p (⋄, ♦). ( C ) Time courses of Sup35p (4 μM) assembly in the absence (•) and in the presence of submolar 0.4 μM (▾, ♦) and equimolar (▴, ▪) concentrations of Ydj1p and Hsp104p, respectively. All assembly reactions were monitored by thioflavin T binding. AU, arbitrary units.

    Techniques Used: Binding Assay

    Effect of Ydj1p and Hsp104p on Sup35p assembly. (A–E and H) Sedimentation analysis of soluble and fibrillar Sup35p (4 μM) ( A ) in the absence and ( B ) the presence of Ydj1p (4 μM), ( C ) Hsp104p (6 μM), ( D ) Hsp104p TRAP (6 μM) and ( E ) Ydj1p and Hsp104p (4 and 6 μM, respectively), in the presence of ATP, GTP and an ATP-regenerating system. Aliquots from each sample were subjected to ultracentrifugation at the time indicated. The resulting supernatants (S) and pellets (P) were analyzed by SDS–PAGE. (F, G) Negative-stained electron micrographs of fibrils made from full-length Sup35p (4 μM) in the presence ( F ) and the absence ( G ) of Hsp104 (12 μM). Bar, 0.5 μm. Length distribution analysis of 400 fibrils made in the presence and 200 fibrils made in the absence of Hsp104p are presented below the electron micrographs. ( H ) SDS–PAGE analysis of supernatants (S) and pellets (P) of insoluble fractions of Sup35p fibrils from the second half of the elongation phase in the presence of Hsp104p and AlF 4 − (S3 and P3), Hsp104p, AlF 4 − and Ssa1p (S4 and P4), Hsp104p, AlF 4 − and Ydj1p (S5 and P5) and Hsp104p, AlF 4 − , Ssa1p and Ydj1p (S6 and P6). Control reactions where Hsp104p alone (S1 and P1) or in the presence of Sup35p fibrils and the absence of AlF 4 − (S2 and P2) were subjected to the same sedimentation conditions are shown.
    Figure Legend Snippet: Effect of Ydj1p and Hsp104p on Sup35p assembly. (A–E and H) Sedimentation analysis of soluble and fibrillar Sup35p (4 μM) ( A ) in the absence and ( B ) the presence of Ydj1p (4 μM), ( C ) Hsp104p (6 μM), ( D ) Hsp104p TRAP (6 μM) and ( E ) Ydj1p and Hsp104p (4 and 6 μM, respectively), in the presence of ATP, GTP and an ATP-regenerating system. Aliquots from each sample were subjected to ultracentrifugation at the time indicated. The resulting supernatants (S) and pellets (P) were analyzed by SDS–PAGE. (F, G) Negative-stained electron micrographs of fibrils made from full-length Sup35p (4 μM) in the presence ( F ) and the absence ( G ) of Hsp104 (12 μM). Bar, 0.5 μm. Length distribution analysis of 400 fibrils made in the presence and 200 fibrils made in the absence of Hsp104p are presented below the electron micrographs. ( H ) SDS–PAGE analysis of supernatants (S) and pellets (P) of insoluble fractions of Sup35p fibrils from the second half of the elongation phase in the presence of Hsp104p and AlF 4 − (S3 and P3), Hsp104p, AlF 4 − and Ssa1p (S4 and P4), Hsp104p, AlF 4 − and Ydj1p (S5 and P5) and Hsp104p, AlF 4 − , Ssa1p and Ydj1p (S6 and P6). Control reactions where Hsp104p alone (S1 and P1) or in the presence of Sup35p fibrils and the absence of AlF 4 − (S2 and P2) were subjected to the same sedimentation conditions are shown.

    Techniques Used: Sedimentation, SDS Page, Staining

    Hsp104p promotes the formation of assembly competent Sup35p oligomeric species. Effect of Hsp104p (6 μM) addition on the nucleation (▪), the elongation (▴) and the steady state (♦) phases of Sup35p (4 μM) assembly. The arrows indicate the time where Hsp104p was added to the assembly reaction, time 0, 20 and 40 h. The assembly of Sup35p in the absence of Hsp104p is shown as a control (•). Assembly was carried out at 10°C and monitored using thioflavin T binding. AU, arbitrary units.
    Figure Legend Snippet: Hsp104p promotes the formation of assembly competent Sup35p oligomeric species. Effect of Hsp104p (6 μM) addition on the nucleation (▪), the elongation (▴) and the steady state (♦) phases of Sup35p (4 μM) assembly. The arrows indicate the time where Hsp104p was added to the assembly reaction, time 0, 20 and 40 h. The assembly of Sup35p in the absence of Hsp104p is shown as a control (•). Assembly was carried out at 10°C and monitored using thioflavin T binding. AU, arbitrary units.

    Techniques Used: Binding Assay

    Seeding properties of Sup35p fibrils assembled in the presence and absence of Hsp104p. ( A ) The elongation rates of increasing concentration of fibrillar Sup35p (0.5, ▪; 1, • and 2 μM, ♦), and that of 1 μM fibrillar Sup35p made in the presence of 6 (○) and 18 μM (⋄) Hsp104p were measured in the presence of 5 μM Sup35p. Control reactions show the assembly of Sup35p in the absence of added seeds (□) and upon addition of 3.6 μM of Hsp104p (▴). The elongation reactions were carried out at 10°C in assembly buffer and were monitored using thioflavin T binding. AU, arbitrary units. ( B ) Linear dependence between the rate of elongation and the amount of fibrils preformed in the absence of Hsp104p, at a constant soluble Sup35p concentration (5 μM). ( C ) Linear dependence between the seeding activity of Sup35p fibrils (1 μM) and the amount of Hsp104p present in the reaction where they were assembled. Panels B and C are based on the experiment described in panel A.
    Figure Legend Snippet: Seeding properties of Sup35p fibrils assembled in the presence and absence of Hsp104p. ( A ) The elongation rates of increasing concentration of fibrillar Sup35p (0.5, ▪; 1, • and 2 μM, ♦), and that of 1 μM fibrillar Sup35p made in the presence of 6 (○) and 18 μM (⋄) Hsp104p were measured in the presence of 5 μM Sup35p. Control reactions show the assembly of Sup35p in the absence of added seeds (□) and upon addition of 3.6 μM of Hsp104p (▴). The elongation reactions were carried out at 10°C in assembly buffer and were monitored using thioflavin T binding. AU, arbitrary units. ( B ) Linear dependence between the rate of elongation and the amount of fibrils preformed in the absence of Hsp104p, at a constant soluble Sup35p concentration (5 μM). ( C ) Linear dependence between the seeding activity of Sup35p fibrils (1 μM) and the amount of Hsp104p present in the reaction where they were assembled. Panels B and C are based on the experiment described in panel A.

    Techniques Used: Concentration Assay, Binding Assay, Activity Assay

    18) Product Images from "Chromatin conformation analysis of primary patient tissue using a low input Hi-C method"

    Article Title: Chromatin conformation analysis of primary patient tissue using a low input Hi-C method

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06961-0

    Compartments, TADs and loops can be detected and are highly reproducible in Low-C data. a Log–log ‘distance decay’ plot for chromosome 1 showing the decrease in contact probability between 50 kb bins with increasing distance for the 1 M and 1 k, as well as the Dixon et al. 15 and Du et al. 12 samples. b AB compartment comparison for chromosome 1 binned at 1 Mb. Contact correlation matrices for the 1 M and 1 k samples (top) and the corresponding first eigenvector (coloured according to the sign of the eigenvector entries) for the samples listed in a are shown on the left. Bottom right shows a scatter plot of first eigenvector values of the 1 M vs. the 1 k sample with Pearson correlation coefficient shown in the plot. Red line indicates identity. c Insulation score 20 comparison for the region on chromosome 13 shown in Fig. 1b . Heatmaps display insulation score values for a range of window sizes, line plots highlight the insulation index for a window size of 250 kb (see panel a for line colours). Next to it is a scatter plot with the Pearson correlation coefficient of the complete insulation index vectors for the 1 k and 1 M samples. Red line indicates identity
    Figure Legend Snippet: Compartments, TADs and loops can be detected and are highly reproducible in Low-C data. a Log–log ‘distance decay’ plot for chromosome 1 showing the decrease in contact probability between 50 kb bins with increasing distance for the 1 M and 1 k, as well as the Dixon et al. 15 and Du et al. 12 samples. b AB compartment comparison for chromosome 1 binned at 1 Mb. Contact correlation matrices for the 1 M and 1 k samples (top) and the corresponding first eigenvector (coloured according to the sign of the eigenvector entries) for the samples listed in a are shown on the left. Bottom right shows a scatter plot of first eigenvector values of the 1 M vs. the 1 k sample with Pearson correlation coefficient shown in the plot. Red line indicates identity. c Insulation score 20 comparison for the region on chromosome 13 shown in Fig. 1b . Heatmaps display insulation score values for a range of window sizes, line plots highlight the insulation index for a window size of 250 kb (see panel a for line colours). Next to it is a scatter plot with the Pearson correlation coefficient of the complete insulation index vectors for the 1 k and 1 M samples. Red line indicates identity

    Techniques Used:

    19) Product Images from "Structure-Function Analysis of DipA, a Francisella tularensis Virulence Factor Required for Intracellular Replication"

    Article Title: Structure-Function Analysis of DipA, a Francisella tularensis Virulence Factor Required for Intracellular Replication

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0067965

    FopA is required for SchuS4 intracellular growth in BMMs and virulence in mice, but not for DipA outer membrane and surface localization. (A) Intracellular growth of SchuS4 and its isogenic ΔFTT1407c mutant in BMMs. BMMs were infected with either strain and CFUs were enumerated at various times p.i. Data are means ± SD from a representative experiment performed in triplicate out of two independent repeats. (B) Intracellular growth of SchuS4, its isogenic Δ fopA and Δ dipAΔfopA mutants and the complemented Δ fopA (p fopA-HA ) strains in BMMs. BMMs were infected with either strain and CFUs were enumerated at various times p.i. Data are means ± SD from a representative experiment performed in triplicate out of two independent repeats. (C) Quantification of bacteria enclosed within LAMP-1-positive phagosomal membranes. BMMs were infected for 1 h with either SchuS4 or its isogenic Δ dipA , Δ fopA and Δ dipAΔfopA mutants. Samples were processed for immunofluorescence labelling of bacteria and LAMP-1-positive membranes. Infected BMMs were scored for number of infected cells with bacteria enclosed within LAMP-1-positive compartments. At least 100 bacteria per experiment were scored for each condition. Data are means ± SD from three independent experiments. (D) Survival curves of BALB/cJ mice infected with SchuS4, SchuS4ΔFTT1407c or SchuS4Δ fopA by intranasal inoculation. Intranasal inocula were 27 (SchuS4), 16 (ΔFTT1407c), and 15 (Δ fopA ) CFUs. (E) Subcellular localization of DipA, FopA, and PdpB from SchuS4Δ fopA (top panels) or SchuS4Δ dipA (bottom panels). Soluble (Sol), inner membrane (IM), and outer membrane (OM) enriched fractions were separated based on Sarkosyl solubility and subjected to immunoblot analysis with antibodies against DipA, FopA, and PdpB. Each fraction was concentrated to the same volume and equal volumes were loaded. (F) Immunoblot analysis of purified surface biotinylated proteins from SchuS4Δ fopA (p dipA-HA ) (top panels) or SchuS4Δ dipA (bottom panels) lysates. DipA-HA was detected using anti-HA antibodies; FopA was detected using anti-FopA antibodies. PdpB and was used as a negative control. Input, untreated (-biotin) and biotinylated (+biotin) samples were processed for CFU enumeration and immunoblotting as described in Materials and Methods. Samples were loaded based on CFU equivalents as follows: 2x10 6 (Input) or 1x10 8 (-/+ biotin) for anti-DipA-HA analysis, 5x10 6 (Input) or 1x10 8 (-/+ biotin) for anti-FopA analysis, 1x10 7 (Input) or 5x10 8 (-/+ biotin) for anti-PdpB analysis.
    Figure Legend Snippet: FopA is required for SchuS4 intracellular growth in BMMs and virulence in mice, but not for DipA outer membrane and surface localization. (A) Intracellular growth of SchuS4 and its isogenic ΔFTT1407c mutant in BMMs. BMMs were infected with either strain and CFUs were enumerated at various times p.i. Data are means ± SD from a representative experiment performed in triplicate out of two independent repeats. (B) Intracellular growth of SchuS4, its isogenic Δ fopA and Δ dipAΔfopA mutants and the complemented Δ fopA (p fopA-HA ) strains in BMMs. BMMs were infected with either strain and CFUs were enumerated at various times p.i. Data are means ± SD from a representative experiment performed in triplicate out of two independent repeats. (C) Quantification of bacteria enclosed within LAMP-1-positive phagosomal membranes. BMMs were infected for 1 h with either SchuS4 or its isogenic Δ dipA , Δ fopA and Δ dipAΔfopA mutants. Samples were processed for immunofluorescence labelling of bacteria and LAMP-1-positive membranes. Infected BMMs were scored for number of infected cells with bacteria enclosed within LAMP-1-positive compartments. At least 100 bacteria per experiment were scored for each condition. Data are means ± SD from three independent experiments. (D) Survival curves of BALB/cJ mice infected with SchuS4, SchuS4ΔFTT1407c or SchuS4Δ fopA by intranasal inoculation. Intranasal inocula were 27 (SchuS4), 16 (ΔFTT1407c), and 15 (Δ fopA ) CFUs. (E) Subcellular localization of DipA, FopA, and PdpB from SchuS4Δ fopA (top panels) or SchuS4Δ dipA (bottom panels). Soluble (Sol), inner membrane (IM), and outer membrane (OM) enriched fractions were separated based on Sarkosyl solubility and subjected to immunoblot analysis with antibodies against DipA, FopA, and PdpB. Each fraction was concentrated to the same volume and equal volumes were loaded. (F) Immunoblot analysis of purified surface biotinylated proteins from SchuS4Δ fopA (p dipA-HA ) (top panels) or SchuS4Δ dipA (bottom panels) lysates. DipA-HA was detected using anti-HA antibodies; FopA was detected using anti-FopA antibodies. PdpB and was used as a negative control. Input, untreated (-biotin) and biotinylated (+biotin) samples were processed for CFU enumeration and immunoblotting as described in Materials and Methods. Samples were loaded based on CFU equivalents as follows: 2x10 6 (Input) or 1x10 8 (-/+ biotin) for anti-DipA-HA analysis, 5x10 6 (Input) or 1x10 8 (-/+ biotin) for anti-FopA analysis, 1x10 7 (Input) or 5x10 8 (-/+ biotin) for anti-PdpB analysis.

    Techniques Used: Mouse Assay, Mutagenesis, Infection, Immunofluorescence, Solubility, Purification, Negative Control

    DipA is exposed to the host cytosol during macrophage infection. (A) Representative confocal micrographs of BMMs infected for 10 h with SchuS4Δ dipA (p dipA-HA ), SchuS4(p iglI-HA ), or SchuS4(p iglA-HA ). Using conditions that permeabilize host plasma membranes but not bacterial membranes (described in Materials and Methods), samples were processed for immunofluorescence labelling of HA-tagged proteins (green) and bacterial LPS (red), and counterstained with DAPI to label DNA (blue). Magnified insets show single channel images of the boxed area. Scale bars, 10 or 2 µm. (B) Quantification of CCF2/AM cleavage in J774A.1 cells that were either uninfected or infected with SchuS4, or SchuS4 expressing C-terminal TEM1 fusions with IglI, IglA or DipA. After 16 h, infected macrophages were loaded with CCF2/AM and analyzed by live cell microscopy for blue fluorescence emission. At least 100 cells were scored per experiment. Data are means ± SD from a representative experiment performed in triplicate out of three independent repeats. Asterisks indicate statistically significant differences compared to uninfected, SchuS4-infected, and SchuS4 expressing IglA-TEM1-infected controls (* P
    Figure Legend Snippet: DipA is exposed to the host cytosol during macrophage infection. (A) Representative confocal micrographs of BMMs infected for 10 h with SchuS4Δ dipA (p dipA-HA ), SchuS4(p iglI-HA ), or SchuS4(p iglA-HA ). Using conditions that permeabilize host plasma membranes but not bacterial membranes (described in Materials and Methods), samples were processed for immunofluorescence labelling of HA-tagged proteins (green) and bacterial LPS (red), and counterstained with DAPI to label DNA (blue). Magnified insets show single channel images of the boxed area. Scale bars, 10 or 2 µm. (B) Quantification of CCF2/AM cleavage in J774A.1 cells that were either uninfected or infected with SchuS4, or SchuS4 expressing C-terminal TEM1 fusions with IglI, IglA or DipA. After 16 h, infected macrophages were loaded with CCF2/AM and analyzed by live cell microscopy for blue fluorescence emission. At least 100 cells were scored per experiment. Data are means ± SD from a representative experiment performed in triplicate out of three independent repeats. Asterisks indicate statistically significant differences compared to uninfected, SchuS4-infected, and SchuS4 expressing IglA-TEM1-infected controls (* P

    Techniques Used: Infection, Immunofluorescence, Expressing, Microscopy, Fluorescence

    DipA is a surface-exposed, membrane-associated protein. (A) Subcellular localization of DipA, FopA, PdpB, GFP, and IglA from GFP-expressing SchuS4. Soluble (Sol), inner membrane (IM), and outer membrane (OM) enriched fractions were separated based on Sarkosyl solubility and subjected to immunoblot analysis with antibodies against DipA, FopA, PdpB, GFP and IglA. Each fraction was concentrated to the same volume and equal volumes were loaded. GFP, PdpB and FopA were used as soluble, inner membrane and outer membrane markers, respectively. (B and C) Immunoblot analysis of purified surface biotinylated proteins from SchuS4Δ dipA (p dipA-HA ) (B) or SchuS4(p iglA-HA ) (C) lysates. DipA-HA and IglA-HA were detected using anti-HA antibodies. FopA was used as a positive control; PdpB and IglA were used as negative controls. Input, untreated (-biotin) and biotinylated (+biotin) samples were processed for CFU enumeration and immunoblotting as described in Materials and Methods. Samples were loaded based on CFU equivalents as follows: 1x10 7 (Input) or 1x10 8 (-/+ biotin) for anti-DipA-HA analysis, 5x10 6 (Input) or 1x10 8 (-/+ biotin) for anti-FopA analysis, 1x10 7 (Input) or 5x10 8 (-/+ biotin) for anti-PdpB analysis, 1x10 7 (Input) or 5x10 8 (-/+ biotin) for anti-IglA analysis, 1x10 7 (Input) or 5x10 8 (-/+ biotin) for anti-IglA-HA analysis.
    Figure Legend Snippet: DipA is a surface-exposed, membrane-associated protein. (A) Subcellular localization of DipA, FopA, PdpB, GFP, and IglA from GFP-expressing SchuS4. Soluble (Sol), inner membrane (IM), and outer membrane (OM) enriched fractions were separated based on Sarkosyl solubility and subjected to immunoblot analysis with antibodies against DipA, FopA, PdpB, GFP and IglA. Each fraction was concentrated to the same volume and equal volumes were loaded. GFP, PdpB and FopA were used as soluble, inner membrane and outer membrane markers, respectively. (B and C) Immunoblot analysis of purified surface biotinylated proteins from SchuS4Δ dipA (p dipA-HA ) (B) or SchuS4(p iglA-HA ) (C) lysates. DipA-HA and IglA-HA were detected using anti-HA antibodies. FopA was used as a positive control; PdpB and IglA were used as negative controls. Input, untreated (-biotin) and biotinylated (+biotin) samples were processed for CFU enumeration and immunoblotting as described in Materials and Methods. Samples were loaded based on CFU equivalents as follows: 1x10 7 (Input) or 1x10 8 (-/+ biotin) for anti-DipA-HA analysis, 5x10 6 (Input) or 1x10 8 (-/+ biotin) for anti-FopA analysis, 1x10 7 (Input) or 5x10 8 (-/+ biotin) for anti-PdpB analysis, 1x10 7 (Input) or 5x10 8 (-/+ biotin) for anti-IglA analysis, 1x10 7 (Input) or 5x10 8 (-/+ biotin) for anti-IglA-HA analysis.

    Techniques Used: Expressing, Solubility, Purification, Positive Control

    The SLR and CC domains of DipA are functionally distinct. (A) Ability of DipA variants to complement the intracellular growth defect of SchuS4Δ dipA . Viable intracellular bacteria were enumerated at 1 h and 16 h p.i. from BMMs infected with SchuS4, SchuS4Δ dipA , or SchuS4Δ dipA expressing HA-tagged DipA variants (DipA-HA, DipAΔSel1ab-HA, DipAΔSel1cd-HA, DipAΔCC-HA, DipACC(AIL 3 D)-HA, or DipACC(LAL 3 D)-HA). Fold change in replication was calculated by comparing CFUs at 16 h p.i. versus 1 h p.i. Data are means ± SD from three independent experiments. Asterisks indicate statistically significant differences compared to SchuS4-infected and SchuS4Δ dipA expressing DipA-HA-infected macrophages (* P
    Figure Legend Snippet: The SLR and CC domains of DipA are functionally distinct. (A) Ability of DipA variants to complement the intracellular growth defect of SchuS4Δ dipA . Viable intracellular bacteria were enumerated at 1 h and 16 h p.i. from BMMs infected with SchuS4, SchuS4Δ dipA , or SchuS4Δ dipA expressing HA-tagged DipA variants (DipA-HA, DipAΔSel1ab-HA, DipAΔSel1cd-HA, DipAΔCC-HA, DipACC(AIL 3 D)-HA, or DipACC(LAL 3 D)-HA). Fold change in replication was calculated by comparing CFUs at 16 h p.i. versus 1 h p.i. Data are means ± SD from three independent experiments. Asterisks indicate statistically significant differences compared to SchuS4-infected and SchuS4Δ dipA expressing DipA-HA-infected macrophages (* P

    Techniques Used: Infection, Expressing

    20) Product Images from "Polymeric Nanocapsules for Vaccine Delivery: Influence of the Polymeric Shell on the Interaction With the Immune System"

    Article Title: Polymeric Nanocapsules for Vaccine Delivery: Influence of the Polymeric Shell on the Interaction With the Immune System

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00791

    Systemic immune response upon intramuscular immunization with rHBsAg PR NCs and PARG NCs. The top graphs represent individual IgG concentrations of anti-HBsAg (expressed in mUI/mL) in each mouse (triangles) and the average of five mice (dash) at 6 weeks (left graph) and 10 weeks (right graph) after the first administration. The number of mice with antibody levels over 100 mIU/mL and the IgG1/IgG2a ratios at different time points are also represented in the lower table.
    Figure Legend Snippet: Systemic immune response upon intramuscular immunization with rHBsAg PR NCs and PARG NCs. The top graphs represent individual IgG concentrations of anti-HBsAg (expressed in mUI/mL) in each mouse (triangles) and the average of five mice (dash) at 6 weeks (left graph) and 10 weeks (right graph) after the first administration. The number of mice with antibody levels over 100 mIU/mL and the IgG1/IgG2a ratios at different time points are also represented in the lower table.

    Techniques Used: Mouse Assay

    Reduction in IκBα expression and activation of mitogen-activated protein kinases [p-extracellular signal-regulated kinase (ERK), p38, and p-SAP/c-Jun NH2-terminal kinases (JNK)] and the nuclear factor κB pathway in Jurkat and Hmy cell lines induced by PR NCs and PARG NCs after 1 h (A) or 3 h (B) of incubation. GAPDH was used as a loading control. White space between lanes shows the cut point in membranes.
    Figure Legend Snippet: Reduction in IκBα expression and activation of mitogen-activated protein kinases [p-extracellular signal-regulated kinase (ERK), p38, and p-SAP/c-Jun NH2-terminal kinases (JNK)] and the nuclear factor κB pathway in Jurkat and Hmy cell lines induced by PR NCs and PARG NCs after 1 h (A) or 3 h (B) of incubation. GAPDH was used as a loading control. White space between lanes shows the cut point in membranes.

    Techniques Used: Expressing, Activation Assay, Incubation

    21) Product Images from "Kaiso Represses the Cell Cycle Gene cyclin D1 via Sequence-Specific and Methyl-CpG-Dependent Mechanisms"

    Article Title: Kaiso Represses the Cell Cycle Gene cyclin D1 via Sequence-Specific and Methyl-CpG-Dependent Mechanisms

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0050398

    Kaiso depletion alters cyclin D1 expression and cell proliferation in HCT116 cells. ( A ) Depletion of endogenous Kaiso with Kaiso-specific siRNA resulted in an ∼ 1.7-fold increase in cyclin D1 protein levels in HCT116 cells. ( B ) Kaiso depletion in HCT116 cells resulted in an ∼ 2-fold increase in cell proliferation.
    Figure Legend Snippet: Kaiso depletion alters cyclin D1 expression and cell proliferation in HCT116 cells. ( A ) Depletion of endogenous Kaiso with Kaiso-specific siRNA resulted in an ∼ 1.7-fold increase in cyclin D1 protein levels in HCT116 cells. ( B ) Kaiso depletion in HCT116 cells resulted in an ∼ 2-fold increase in cell proliferation.

    Techniques Used: Expressing

    22) Product Images from "Critical Role of FLRT1 Phosphorylation in the Interdependent Regulation of FLRT1 Function and FGF Receptor Signalling"

    Article Title: Critical Role of FLRT1 Phosphorylation in the Interdependent Regulation of FLRT1 Function and FGF Receptor Signalling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0010264

    Co-localization of FLRT1 and FGFR1. A) Immunofluorescent staining of Cos-7 cells co-transfected with plasmids encoding FGFR1 and 3'HA-tagged FLRT1 Cells were stained with anti-FGFR1 (green) and anti-HA (FLRT1 -red) Merged images show areas of co-localisation in yellow Images (in section) were taken with a confocal microscope and are representative cells from 11 total fields of cells B) HEK 293T cells were co-transfected with FGFR1 and either control vector (pcDNA31) or FLRT1 (FLRT-HA) with or without stimulation with FGF2 (20ng/ml) in the presence of heparin (10mg/ml) for 30 min Anti-HA immunoprecipitation was performed on whole cell lysate which was subjected to western blot analysis with anti-phosphotyrosine (IP: HA, Blot: pY) to identify phosphorylated FLRT1 (pFLRT1) Phosphorylated FGFR1 (pFGFR1) was co-immunoprecipitated with FLRT1 The whole cell lysate (WCL) was probed for both FGFR1 (Blot: anti-FGFR1) and FLRT1 (Blot: anti-HA) expression Data is representative of at least 4 independent experiments.
    Figure Legend Snippet: Co-localization of FLRT1 and FGFR1. A) Immunofluorescent staining of Cos-7 cells co-transfected with plasmids encoding FGFR1 and 3'HA-tagged FLRT1 Cells were stained with anti-FGFR1 (green) and anti-HA (FLRT1 -red) Merged images show areas of co-localisation in yellow Images (in section) were taken with a confocal microscope and are representative cells from 11 total fields of cells B) HEK 293T cells were co-transfected with FGFR1 and either control vector (pcDNA31) or FLRT1 (FLRT-HA) with or without stimulation with FGF2 (20ng/ml) in the presence of heparin (10mg/ml) for 30 min Anti-HA immunoprecipitation was performed on whole cell lysate which was subjected to western blot analysis with anti-phosphotyrosine (IP: HA, Blot: pY) to identify phosphorylated FLRT1 (pFLRT1) Phosphorylated FGFR1 (pFGFR1) was co-immunoprecipitated with FLRT1 The whole cell lysate (WCL) was probed for both FGFR1 (Blot: anti-FGFR1) and FLRT1 (Blot: anti-HA) expression Data is representative of at least 4 independent experiments.

    Techniques Used: Staining, Transfection, Microscopy, Plasmid Preparation, Immunoprecipitation, Western Blot, Expressing

    FLRT1 is not a SFK substrate but phosphorylation is FGFR-and SFK-dependent. A) HEK 293T cells were transfected with control (pcDNA31) or FGFR1 and a panel of either full-length FLRT1-HA or tyrosine substitution contructs as indicated (see Materials Methods ) One sample was pre-treated with FGFR kinase inhibitor (SU5402, 50mM, 1 hr) where indicated Cell lysates were immunoprecipitated with anti-HA and subsequently blotted with anti-phosphotyrosine (IP: HA, Blot: pY) or anti-HA (IP: HA, Blot: HA) to examine phosphorylated FLRT1HA levels (pFLRT1) or total immunoprecipitated FLRT1-HA levels (FLRT1), respectively Whole cell lysate (WCL) fractions were probed with anti-FGFR1 (Blot: FGFR) to control for protein expression B) HEK 293T cells were transfected with pcDNA31, FGFR1 and FLRT1-HA as indicated Cells were pre-incubated (1hr) with pharmacological inhibitors (SU5402, 50mM; SU6656, 20mM) Cell lysates were immunoprecipitated with anti-HA and subsequently blotted with anti-phosphotyrosine (IP: HA, Blot: pY) for pFLRT1 and anti-HA (IP: HA, Blot: HA) for FLRT1 WCL fractions were probed with anti-FGFR1 (Blot: FGFR), anti-phospho-ERK (Blot: pERK) or anti-ERK (Blot: ERK) Data in A) and B) are representative of ≥3 independent experiments Densitometric analysis (mean ± sem, n = 3) is the ratio of pFLRT1:FLRT1 and normalised to FLRT1 phosphorylation in the absence of inhibitor in both cases (**p
    Figure Legend Snippet: FLRT1 is not a SFK substrate but phosphorylation is FGFR-and SFK-dependent. A) HEK 293T cells were transfected with control (pcDNA31) or FGFR1 and a panel of either full-length FLRT1-HA or tyrosine substitution contructs as indicated (see Materials Methods ) One sample was pre-treated with FGFR kinase inhibitor (SU5402, 50mM, 1 hr) where indicated Cell lysates were immunoprecipitated with anti-HA and subsequently blotted with anti-phosphotyrosine (IP: HA, Blot: pY) or anti-HA (IP: HA, Blot: HA) to examine phosphorylated FLRT1HA levels (pFLRT1) or total immunoprecipitated FLRT1-HA levels (FLRT1), respectively Whole cell lysate (WCL) fractions were probed with anti-FGFR1 (Blot: FGFR) to control for protein expression B) HEK 293T cells were transfected with pcDNA31, FGFR1 and FLRT1-HA as indicated Cells were pre-incubated (1hr) with pharmacological inhibitors (SU5402, 50mM; SU6656, 20mM) Cell lysates were immunoprecipitated with anti-HA and subsequently blotted with anti-phosphotyrosine (IP: HA, Blot: pY) for pFLRT1 and anti-HA (IP: HA, Blot: HA) for FLRT1 WCL fractions were probed with anti-FGFR1 (Blot: FGFR), anti-phospho-ERK (Blot: pERK) or anti-ERK (Blot: ERK) Data in A) and B) are representative of ≥3 independent experiments Densitometric analysis (mean ± sem, n = 3) is the ratio of pFLRT1:FLRT1 and normalised to FLRT1 phosphorylation in the absence of inhibitor in both cases (**p

    Techniques Used: Transfection, Immunoprecipitation, Expressing, Incubation

    23) Product Images from "A map of the phosphoproteomic alterations that occur after a bout of maximal‐intensity contractions"

    Article Title: A map of the phosphoproteomic alterations that occur after a bout of maximal‐intensity contractions

    Journal: The Journal of Physiology

    doi: 10.1113/JP273904

    Validation of maximal‐intensity contraction‐induced signalling events and centrifugation methods prior to 6‐plex TMT sample preparation Tibialis anterior muscles were subjected to a bout of maximal‐intensity contractions (MIC) or the control condition. In the first workflow (black) the muscles were homogenized in a urea lysis buffer, while in the second workflow (blue) the muscles were homogenized in Triton X‐100 lysis buffer and then centrifuged to separate the proteome into pellet and supernatant fractions. Prior to MS analysis, all samples were subjected to Western blotting to confirm that the bout of MIC had induced the activation of signalling through mTOR [P‐p70S6K(389)] and p38 [P‐p38(180/2)]. Western blotting of actin, as well as Coomassie blue staining of all proteins, was also performed to illustrate the differences in the proteome of the pellet and supernatant fractions.
    Figure Legend Snippet: Validation of maximal‐intensity contraction‐induced signalling events and centrifugation methods prior to 6‐plex TMT sample preparation Tibialis anterior muscles were subjected to a bout of maximal‐intensity contractions (MIC) or the control condition. In the first workflow (black) the muscles were homogenized in a urea lysis buffer, while in the second workflow (blue) the muscles were homogenized in Triton X‐100 lysis buffer and then centrifuged to separate the proteome into pellet and supernatant fractions. Prior to MS analysis, all samples were subjected to Western blotting to confirm that the bout of MIC had induced the activation of signalling through mTOR [P‐p70S6K(389)] and p38 [P‐p38(180/2)]. Western blotting of actin, as well as Coomassie blue staining of all proteins, was also performed to illustrate the differences in the proteome of the pellet and supernatant fractions.

    Techniques Used: Centrifugation, Sample Prep, Lysis, Mass Spectrometry, Western Blot, Activation Assay, Staining

    Overall analysis of the effect of maximal‐intensity contractions on phosphopeptide abundance In two independent experiments, mouse tibialis anterior muscles were subjected to a bout of maximal‐intensity contractions (MIC) or the control condition and then prepared for MS analysis. In the first experiment ( n  = 3 per group) the samples were homogenized in a urea lysis buffer, while in the second experiment ( n  = 3 per group) the samples were homogenized in a Triton X‐100 lysis buffer and then centrifuged to separate the proteome into pellet and supernatant fractions.  A , the results from the first and second experiment were merged and the fold‐changes (MIC/Control) of the phosphopeptides were log2 transformed and plotted  versus  their corresponding –log10  P ‐values. Phosphopeptides that revealed major MIC‐induced alterations ( >  1.5‐fold,  P  ≤ 0.05) are highlighted in blue (increased) or red (decreased).  B , Western blot analysis was performed on specific phosphorylation sites which were concluded by MS to have experienced no change (threonine 287 on calmodulin kinase 2 (CAMK2β)), an increase (serine 86 on heat shock protein 27 (HSP27)), or a decrease (serine 229 on glucocorticoid receptor (GR)) in phosphorylation. Western blot analysis for actin was used to verify equal loading of protein in all lanes.
    Figure Legend Snippet: Overall analysis of the effect of maximal‐intensity contractions on phosphopeptide abundance In two independent experiments, mouse tibialis anterior muscles were subjected to a bout of maximal‐intensity contractions (MIC) or the control condition and then prepared for MS analysis. In the first experiment ( n  = 3 per group) the samples were homogenized in a urea lysis buffer, while in the second experiment ( n  = 3 per group) the samples were homogenized in a Triton X‐100 lysis buffer and then centrifuged to separate the proteome into pellet and supernatant fractions. A , the results from the first and second experiment were merged and the fold‐changes (MIC/Control) of the phosphopeptides were log2 transformed and plotted versus their corresponding –log10 P ‐values. Phosphopeptides that revealed major MIC‐induced alterations ( >  1.5‐fold, P  ≤ 0.05) are highlighted in blue (increased) or red (decreased). B , Western blot analysis was performed on specific phosphorylation sites which were concluded by MS to have experienced no change (threonine 287 on calmodulin kinase 2 (CAMK2β)), an increase (serine 86 on heat shock protein 27 (HSP27)), or a decrease (serine 229 on glucocorticoid receptor (GR)) in phosphorylation. Western blot analysis for actin was used to verify equal loading of protein in all lanes.

    Techniques Used: Mass Spectrometry, Lysis, Transformation Assay, Western Blot

    Comparison of the phosphopeptides and proteins that were identified with the different sample preparation methods Mouse tibialis anterior muscles were subjected to a bout of maximal‐intensity contractions (MIC) or the control condition and then prepared for MS analysis. Samples in experiment 1 ( n  = 3 per group) were prepared via homogenization in a urea lysis buffer, while samples in experiment 2 ( n  = 3 per group) were prepared via homogenization in a Triton X‐100 buffer and then further separated by centrifugation into supernatant and pellet fractions. The differently prepared samples/fractions from each experiment were subjected to separate 6‐plex TMT MS analyses. Venn diagrams were generated to compare the unique phosphopeptide ( A ) and protein ( B ) populations that were quantified in experiment 1 (grey) and the supernatant (red) and pellet (blue) fractions in experiment 2. Intermediately shaded regions indicate the phosphopeptides or proteins that were observed in multiple experiments.
    Figure Legend Snippet: Comparison of the phosphopeptides and proteins that were identified with the different sample preparation methods Mouse tibialis anterior muscles were subjected to a bout of maximal‐intensity contractions (MIC) or the control condition and then prepared for MS analysis. Samples in experiment 1 ( n  = 3 per group) were prepared via homogenization in a urea lysis buffer, while samples in experiment 2 ( n  = 3 per group) were prepared via homogenization in a Triton X‐100 buffer and then further separated by centrifugation into supernatant and pellet fractions. The differently prepared samples/fractions from each experiment were subjected to separate 6‐plex TMT MS analyses. Venn diagrams were generated to compare the unique phosphopeptide ( A ) and protein ( B ) populations that were quantified in experiment 1 (grey) and the supernatant (red) and pellet (blue) fractions in experiment 2. Intermediately shaded regions indicate the phosphopeptides or proteins that were observed in multiple experiments.

    Techniques Used: Sample Prep, Mass Spectrometry, Homogenization, Lysis, Centrifugation, Generated

    24) Product Images from "Natural variation in the histone demethylase, KDM4C, influences expression levels of specific genes including those that affect cell growth"

    Article Title: Natural variation in the histone demethylase, KDM4C, influences expression levels of specific genes including those that affect cell growth

    Journal: Genome Research

    doi: 10.1101/gr.156141.113

    Chromosome 9p24 that includes KDM4C is a gene expression regulatory region. ( A ) Linkage plots for four gene expression phenotypes that show significant linkage to chromosome 9p24. ( B ) Results from QTDT and population association with SNPs in expressed
    Figure Legend Snippet: Chromosome 9p24 that includes KDM4C is a gene expression regulatory region. ( A ) Linkage plots for four gene expression phenotypes that show significant linkage to chromosome 9p24. ( B ) Results from QTDT and population association with SNPs in expressed

    Techniques Used: Expressing

    KDM4C expression influences target gene expression levels. ( A ) Chromatin immunoprecipitation (ChIP) for KDM4C at target gene promoters in B cells from three individuals with high and three individuals with low KDM4C expression. The negative controls are
    Figure Legend Snippet: KDM4C expression influences target gene expression levels. ( A ) Chromatin immunoprecipitation (ChIP) for KDM4C at target gene promoters in B cells from three individuals with high and three individuals with low KDM4C expression. The negative controls are

    Techniques Used: Expressing, Chromatin Immunoprecipitation

    Molecular validation of KDM4C target genes in B cells and primary fibroblasts. ( A ) B cells from four individuals were treated with siRNA targeting KDM4C or a control siRNA. ( Top ) qRT-PCR data confirming KDM4C depletion at the transcript level; ( bottom
    Figure Legend Snippet: Molecular validation of KDM4C target genes in B cells and primary fibroblasts. ( A ) B cells from four individuals were treated with siRNA targeting KDM4C or a control siRNA. ( Top ) qRT-PCR data confirming KDM4C depletion at the transcript level; ( bottom

    Techniques Used: Quantitative RT-PCR

    Cis -regulation of KDM4C . ( A ) KDM4C expression levels in cultured B cells of 24 unrelated individuals (open circles) and primary B cells of nine unrelated individuals (filled circles) are shown relative to the individual with the lowest expression, as
    Figure Legend Snippet: Cis -regulation of KDM4C . ( A ) KDM4C expression levels in cultured B cells of 24 unrelated individuals (open circles) and primary B cells of nine unrelated individuals (filled circles) are shown relative to the individual with the lowest expression, as

    Techniques Used: Expressing, Cell Culture

    The 3′ regulatory region of KDM4C shows enhancer activity. ( A ) The black bar above the KDM4C gene schematic represents the region containing SNPs with significant association to KDM4C expression; the position of SNP rs7868863 is marked. ( Bottom
    Figure Legend Snippet: The 3′ regulatory region of KDM4C shows enhancer activity. ( A ) The black bar above the KDM4C gene schematic represents the region containing SNPs with significant association to KDM4C expression; the position of SNP rs7868863 is marked. ( Bottom

    Techniques Used: Activity Assay, Expressing

    KDM4C overexpression accelerates cell growth. ( A ) Overexpression of KDM4C in primary fibroblasts was confirmed by qRT-PCR and Western blot relative to control cells. ( B ) Target gene expression changes following KDM4C overexpression in primary fibroblasts
    Figure Legend Snippet: KDM4C overexpression accelerates cell growth. ( A ) Overexpression of KDM4C in primary fibroblasts was confirmed by qRT-PCR and Western blot relative to control cells. ( B ) Target gene expression changes following KDM4C overexpression in primary fibroblasts

    Techniques Used: Over Expression, Quantitative RT-PCR, Western Blot, Expressing

    KDM4C expression level in cancer cells. ( A ) Average KDM4C expression level in cancer tissues relative to matching normal tissues, by qRT-PCR. ( B ) Ratios of KDM4C expression levels in cancers and the matching controls in seven tissues by our qRT-PCR compared
    Figure Legend Snippet: KDM4C expression level in cancer cells. ( A ) Average KDM4C expression level in cancer tissues relative to matching normal tissues, by qRT-PCR. ( B ) Ratios of KDM4C expression levels in cancers and the matching controls in seven tissues by our qRT-PCR compared

    Techniques Used: Expressing, Quantitative RT-PCR

    KDM4C expression is associated with cell growth in B cells. ( A ) Growth curve of B cells from five individuals with high or low KDM4C expression ( P = 3 × 10 −6 , ANOVA). ( B ) BrdU incorporation in B cells from six individuals with high or
    Figure Legend Snippet: KDM4C expression is associated with cell growth in B cells. ( A ) Growth curve of B cells from five individuals with high or low KDM4C expression ( P = 3 × 10 −6 , ANOVA). ( B ) BrdU incorporation in B cells from six individuals with high or

    Techniques Used: Expressing, BrdU Incorporation Assay

    25) Product Images from "Natural variation in the histone demethylase, KDM4C, influences expression levels of specific genes including those that affect cell growth"

    Article Title: Natural variation in the histone demethylase, KDM4C, influences expression levels of specific genes including those that affect cell growth

    Journal: Genome Research

    doi: 10.1101/gr.156141.113

    Chromosome 9p24 that includes KDM4C is a gene expression regulatory region. ( A ) Linkage plots for four gene expression phenotypes that show significant linkage to chromosome 9p24. ( B ) Results from QTDT and population association with SNPs in expressed
    Figure Legend Snippet: Chromosome 9p24 that includes KDM4C is a gene expression regulatory region. ( A ) Linkage plots for four gene expression phenotypes that show significant linkage to chromosome 9p24. ( B ) Results from QTDT and population association with SNPs in expressed

    Techniques Used: Expressing

    KDM4C expression influences target gene expression levels. ( A ) Chromatin immunoprecipitation (ChIP) for KDM4C at target gene promoters in B cells from three individuals with high and three individuals with low KDM4C expression. The negative controls are
    Figure Legend Snippet: KDM4C expression influences target gene expression levels. ( A ) Chromatin immunoprecipitation (ChIP) for KDM4C at target gene promoters in B cells from three individuals with high and three individuals with low KDM4C expression. The negative controls are

    Techniques Used: Expressing, Chromatin Immunoprecipitation

    Molecular validation of KDM4C target genes in B cells and primary fibroblasts. ( A ) B cells from four individuals were treated with siRNA targeting KDM4C or a control siRNA. ( Top ) qRT-PCR data confirming KDM4C depletion at the transcript level; ( bottom
    Figure Legend Snippet: Molecular validation of KDM4C target genes in B cells and primary fibroblasts. ( A ) B cells from four individuals were treated with siRNA targeting KDM4C or a control siRNA. ( Top ) qRT-PCR data confirming KDM4C depletion at the transcript level; ( bottom

    Techniques Used: Quantitative RT-PCR

    Cis -regulation of KDM4C . ( A ) KDM4C expression levels in cultured B cells of 24 unrelated individuals (open circles) and primary B cells of nine unrelated individuals (filled circles) are shown relative to the individual with the lowest expression, as
    Figure Legend Snippet: Cis -regulation of KDM4C . ( A ) KDM4C expression levels in cultured B cells of 24 unrelated individuals (open circles) and primary B cells of nine unrelated individuals (filled circles) are shown relative to the individual with the lowest expression, as

    Techniques Used: Expressing, Cell Culture

    The 3′ regulatory region of KDM4C shows enhancer activity. ( A ) The black bar above the KDM4C gene schematic represents the region containing SNPs with significant association to KDM4C expression; the position of SNP rs7868863 is marked. ( Bottom
    Figure Legend Snippet: The 3′ regulatory region of KDM4C shows enhancer activity. ( A ) The black bar above the KDM4C gene schematic represents the region containing SNPs with significant association to KDM4C expression; the position of SNP rs7868863 is marked. ( Bottom

    Techniques Used: Activity Assay, Expressing

    KDM4C overexpression accelerates cell growth. ( A ) Overexpression of KDM4C in primary fibroblasts was confirmed by qRT-PCR and Western blot relative to control cells. ( B ) Target gene expression changes following KDM4C overexpression in primary fibroblasts
    Figure Legend Snippet: KDM4C overexpression accelerates cell growth. ( A ) Overexpression of KDM4C in primary fibroblasts was confirmed by qRT-PCR and Western blot relative to control cells. ( B ) Target gene expression changes following KDM4C overexpression in primary fibroblasts

    Techniques Used: Over Expression, Quantitative RT-PCR, Western Blot, Expressing

    KDM4C expression level in cancer cells. ( A ) Average KDM4C expression level in cancer tissues relative to matching normal tissues, by qRT-PCR. ( B ) Ratios of KDM4C expression levels in cancers and the matching controls in seven tissues by our qRT-PCR compared
    Figure Legend Snippet: KDM4C expression level in cancer cells. ( A ) Average KDM4C expression level in cancer tissues relative to matching normal tissues, by qRT-PCR. ( B ) Ratios of KDM4C expression levels in cancers and the matching controls in seven tissues by our qRT-PCR compared

    Techniques Used: Expressing, Quantitative RT-PCR

    KDM4C expression is associated with cell growth in B cells. ( A ) Growth curve of B cells from five individuals with high or low KDM4C expression ( P = 3 × 10 −6 , ANOVA). ( B ) BrdU incorporation in B cells from six individuals with high or
    Figure Legend Snippet: KDM4C expression is associated with cell growth in B cells. ( A ) Growth curve of B cells from five individuals with high or low KDM4C expression ( P = 3 × 10 −6 , ANOVA). ( B ) BrdU incorporation in B cells from six individuals with high or

    Techniques Used: Expressing, BrdU Incorporation Assay

    26) Product Images from "Progesterone Receptor Membrane Component 1 Is a Functional Part of the Glucagon-like Peptide-1 (GLP-1) Receptor Complex in Pancreatic β Cells *"

    Article Title: Progesterone Receptor Membrane Component 1 Is a Functional Part of the Glucagon-like Peptide-1 (GLP-1) Receptor Complex in Pancreatic β Cells *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.M114.040196

    The interaction between GLP-1R and selected interactors as assessed by immunofluorescence. A , immunofluorescence images showing colocalization of selected interactors (red) and GLP-1R (green) in MIN6 cells. NRGN (neurogranin), a known non-interactive
    Figure Legend Snippet: The interaction between GLP-1R and selected interactors as assessed by immunofluorescence. A , immunofluorescence images showing colocalization of selected interactors (red) and GLP-1R (green) in MIN6 cells. NRGN (neurogranin), a known non-interactive

    Techniques Used: Immunofluorescence

    AP-MS was used to profile potential GLP-1R interactors and their functional clusters in CHO cells ( A ) and MIN6 cells ( B ). The potential GLP-1R binding proteins include those exclusively identified in the unliganded cells (green), in the liganded cells
    Figure Legend Snippet: AP-MS was used to profile potential GLP-1R interactors and their functional clusters in CHO cells ( A ) and MIN6 cells ( B ). The potential GLP-1R binding proteins include those exclusively identified in the unliganded cells (green), in the liganded cells

    Techniques Used: Mass Spectrometry, Functional Assay, Binding Assay

    Discovery of novel GLP-1R interactors. A , flow chart showing the AP-MS strategy used to identify novel GLP-1R interactors. Non-transfected (NT) CHO/MIN6 cells were used as the negative control group. CHO/MIN6 cells expressing GLP-1R-Flag were used with
    Figure Legend Snippet: Discovery of novel GLP-1R interactors. A , flow chart showing the AP-MS strategy used to identify novel GLP-1R interactors. Non-transfected (NT) CHO/MIN6 cells were used as the negative control group. CHO/MIN6 cells expressing GLP-1R-Flag were used with

    Techniques Used: Flow Cytometry, Mass Spectrometry, Transfection, Negative Control, Expressing

    Validation of selected GLP-1R interactors by co-IP and western blot. A , anti-Flag co-IP and anti-HA western blot of selected interactors from MIN6 cells co-transfected with GLP-1R-Flag and interactor-HA ( left panel) or transfected with interactor-HA alone
    Figure Legend Snippet: Validation of selected GLP-1R interactors by co-IP and western blot. A , anti-Flag co-IP and anti-HA western blot of selected interactors from MIN6 cells co-transfected with GLP-1R-Flag and interactor-HA ( left panel) or transfected with interactor-HA alone

    Techniques Used: Co-Immunoprecipitation Assay, Western Blot, Transfection

    Modulation of PGRMC1 activity affects insulin secretion in INS1 832/3 cells ( A ), MIN6 cells ( B ), and isolated primary mouse islets ( C ). The PGRMC1 surface ligand P4-BSA (1 μ m ) and AG205 (5 μ m ) were used to activate and inhibit PGRMC1,
    Figure Legend Snippet: Modulation of PGRMC1 activity affects insulin secretion in INS1 832/3 cells ( A ), MIN6 cells ( B ), and isolated primary mouse islets ( C ). The PGRMC1 surface ligand P4-BSA (1 μ m ) and AG205 (5 μ m ) were used to activate and inhibit PGRMC1,

    Techniques Used: Activity Assay, Isolation

    27) Product Images from "Extracellular vesicles in DLBCL provide abundant clues to aberrant transcriptional programming and genomic alterations"

    Article Title: Extracellular vesicles in DLBCL provide abundant clues to aberrant transcriptional programming and genomic alterations

    Journal: Blood

    doi: 10.1182/blood-2017-12-821843

    RNA species found in EVs and cells. (A) DLBCL cell lines OCI-Ly3, HBL1, TMD8, OCI-Ly1, and OCI-Ly7, (B) EVs from DLBCL cell lines OCI-Ly3, HBL1, TMD8, OCI-Ly1, and OCI-Ly7, (C) whole cell DLBCL primary samples DLBCL1, DLBCL2, DLBCL3, DLBCL4, DLBCL5, (D) EVs from DLBCL primary sample DLBCL6, (E) whole cell normal B cells NB1, NB2, and NB3, and (F) EVs from normal B cells B2. EVs contain a variety of RNA species with enrichment for noncoding RNAs, long intergenic noncoding RNAs (lincRNAs), snoRNAs, and snRNAs. Quantities are displayed as the sum of average transcripts per million (TPM) per RNA class. rRNA, ribosomal RNA.
    Figure Legend Snippet: RNA species found in EVs and cells. (A) DLBCL cell lines OCI-Ly3, HBL1, TMD8, OCI-Ly1, and OCI-Ly7, (B) EVs from DLBCL cell lines OCI-Ly3, HBL1, TMD8, OCI-Ly1, and OCI-Ly7, (C) whole cell DLBCL primary samples DLBCL1, DLBCL2, DLBCL3, DLBCL4, DLBCL5, (D) EVs from DLBCL primary sample DLBCL6, (E) whole cell normal B cells NB1, NB2, and NB3, and (F) EVs from normal B cells B2. EVs contain a variety of RNA species with enrichment for noncoding RNAs, long intergenic noncoding RNAs (lincRNAs), snoRNAs, and snRNAs. Quantities are displayed as the sum of average transcripts per million (TPM) per RNA class. rRNA, ribosomal RNA.

    Techniques Used:

    EV exchange by DLBCL cell lines and uptake by normal tonsillar cells. (A) Positive SYTO RNA fluorescent stain of supernatant from recipient OCI-Ly3 cells previously incubated with EVs from stained donor OCI-Ly1 cells (left panel) and positive stain for the general exosome marker CD63 (right panel), indicating uptake of RNAs from stained OCI-Ly1 donor cells. (B) Control negative SYTO RNA fluorescent staining of supernatant from recipient OCI-Ly3 cells incubated with EVs of unstained OCI-Ly1 donor cells (left panel), and positive staining for CD63 (right panel). (C) Positive SYTO RNA fluorescent stain in recipient normal tonsillar lymphocytes incubated with EVs isolated from previously stained donor OCI-Ly1 (left panel) or HBL1 (right panel).
    Figure Legend Snippet: EV exchange by DLBCL cell lines and uptake by normal tonsillar cells. (A) Positive SYTO RNA fluorescent stain of supernatant from recipient OCI-Ly3 cells previously incubated with EVs from stained donor OCI-Ly1 cells (left panel) and positive stain for the general exosome marker CD63 (right panel), indicating uptake of RNAs from stained OCI-Ly1 donor cells. (B) Control negative SYTO RNA fluorescent staining of supernatant from recipient OCI-Ly3 cells incubated with EVs of unstained OCI-Ly1 donor cells (left panel), and positive staining for CD63 (right panel). (C) Positive SYTO RNA fluorescent stain in recipient normal tonsillar lymphocytes incubated with EVs isolated from previously stained donor OCI-Ly1 (left panel) or HBL1 (right panel).

    Techniques Used: Staining, Incubation, Marker, Isolation

    28) Product Images from "Glutamate reduces glucose utilization while concomitantly enhancing AQP9 and MCT2 expression in cultured rat hippocampal neurons"

    Article Title: Glutamate reduces glucose utilization while concomitantly enhancing AQP9 and MCT2 expression in cultured rat hippocampal neurons

    Journal: Frontiers in Neuroscience

    doi: 10.3389/fnins.2014.00246

    Colocalization and interaction between AQP9 and MCT2 in cultured rat hippocampal neurons (A) Double immunofluorescence labeling for AQP9 (in green) and MCT2 (in red) performed in cultured hippocampal neurons . Colocalization between AQP9 and MCT2 appears as yellow on the merged image (Merge). Nuclei (in blue) were labeled using Hoechst staining. Immunofluorescence was visualized using confocal microscopy with appropriate filters. (B) Western blot for AQP9 in cultured hippocampal neuron protein extract after immunoprecipitation with anti-MCT2 antibody (IP MCT2) or in total protein extract. This experiment was repeated twice from separate cultures with similar results.
    Figure Legend Snippet: Colocalization and interaction between AQP9 and MCT2 in cultured rat hippocampal neurons (A) Double immunofluorescence labeling for AQP9 (in green) and MCT2 (in red) performed in cultured hippocampal neurons . Colocalization between AQP9 and MCT2 appears as yellow on the merged image (Merge). Nuclei (in blue) were labeled using Hoechst staining. Immunofluorescence was visualized using confocal microscopy with appropriate filters. (B) Western blot for AQP9 in cultured hippocampal neuron protein extract after immunoprecipitation with anti-MCT2 antibody (IP MCT2) or in total protein extract. This experiment was repeated twice from separate cultures with similar results.

    Techniques Used: Cell Culture, Immunofluorescence, Labeling, Staining, Confocal Microscopy, Western Blot, Immunoprecipitation

    29) Product Images from "RNF185 Is a Novel E3 Ligase of Endoplasmic Reticulum-associated Degradation (ERAD) That Targets Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) *"

    Article Title: RNF185 Is a Novel E3 Ligase of Endoplasmic Reticulum-associated Degradation (ERAD) That Targets Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.470500

    RNF185 is an RNF5 homolog conserved in higher eukaryotes. Amino acid sequence alignment is shown for human (GI, 45708382) and mouse (GI, 15928691) RNF185 with their human (GI, 5902054), mouse (GI, 9507059), and Caenorhabditis elegans RNF5 (GI, 3874385)
    Figure Legend Snippet: RNF185 is an RNF5 homolog conserved in higher eukaryotes. Amino acid sequence alignment is shown for human (GI, 45708382) and mouse (GI, 15928691) RNF185 with their human (GI, 5902054), mouse (GI, 9507059), and Caenorhabditis elegans RNF5 (GI, 3874385)

    Techniques Used: Sequencing

    RNF185 interacts with ERAD components and is induced by UPR. A , RNF185 interacts with Derlin-1 and Erlin2. HEK293T cells were transfected with the control vector, FLAG-RNF185, or FLAG RNF5 (0.5 μg of plasmid per well of a 6-well plate). 24 h post-transfection,
    Figure Legend Snippet: RNF185 interacts with ERAD components and is induced by UPR. A , RNF185 interacts with Derlin-1 and Erlin2. HEK293T cells were transfected with the control vector, FLAG-RNF185, or FLAG RNF5 (0.5 μg of plasmid per well of a 6-well plate). 24 h post-transfection,

    Techniques Used: Transfection, Plasmid Preparation

    RNF185 induces the ubiquitin-proteasome-dependent degradation of CFTR proteins. A , RNF185 overexpression decreases the steady-state levels of WT CFTR and CFTRΔF508. Cells were co-transfected with control vector or increasing amounts of FLAG-RNF185
    Figure Legend Snippet: RNF185 induces the ubiquitin-proteasome-dependent degradation of CFTR proteins. A , RNF185 overexpression decreases the steady-state levels of WT CFTR and CFTRΔF508. Cells were co-transfected with control vector or increasing amounts of FLAG-RNF185

    Techniques Used: Over Expression, Transfection, Plasmid Preparation

    RNF185 is a novel ubiquitously expressed E3 ligase. A , expression of RNF185 in mouse tissues. Total RNAs were purified from WT mouse tissues and were retrotranscribed for quantitative-PCR analysis using RNF185 -specific primers. PPIA1 and 18 S RNA were
    Figure Legend Snippet: RNF185 is a novel ubiquitously expressed E3 ligase. A , expression of RNF185 in mouse tissues. Total RNAs were purified from WT mouse tissues and were retrotranscribed for quantitative-PCR analysis using RNF185 -specific primers. PPIA1 and 18 S RNA were

    Techniques Used: Expressing, Purification, Real-time Polymerase Chain Reaction

    RNF185 targets CFTRΔF508 to co-translational degradation. A , measure of CFTRΔF508 labeling rates upon RNF185 overexpression. Cells were co-transfected with CFTRΔF508-HA together with RNF185 or the corresponding control vector.
    Figure Legend Snippet: RNF185 targets CFTRΔF508 to co-translational degradation. A , measure of CFTRΔF508 labeling rates upon RNF185 overexpression. Cells were co-transfected with CFTRΔF508-HA together with RNF185 or the corresponding control vector.

    Techniques Used: Labeling, Over Expression, Transfection, Plasmid Preparation

    Combined depletion of RNF185 and RNF5 synergistically blocks CFTRΔF508 degradation. A , analysis of CFTRΔF508 turnover upon combined RNF185 and RNF5 knockdown. HEK293 cells stably expressing a control shRNA or an shRNA sequence targeting
    Figure Legend Snippet: Combined depletion of RNF185 and RNF5 synergistically blocks CFTRΔF508 degradation. A , analysis of CFTRΔF508 turnover upon combined RNF185 and RNF5 knockdown. HEK293 cells stably expressing a control shRNA or an shRNA sequence targeting

    Techniques Used: Stable Transfection, Expressing, shRNA, Sequencing

    Analysis of CFTRΔF508 degradation by cycloheximide chase. A , CHX chase analysis of CFTRΔF508 upon RNF185 expression. HEK293T cells were co-transfected with CFTRΔF508-HA together with a control vector or vector expressing RNF185
    Figure Legend Snippet: Analysis of CFTRΔF508 degradation by cycloheximide chase. A , CHX chase analysis of CFTRΔF508 upon RNF185 expression. HEK293T cells were co-transfected with CFTRΔF508-HA together with a control vector or vector expressing RNF185

    Techniques Used: Expressing, Transfection, Plasmid Preparation

    RNF185 is an ER-localized E3 ligase. A , co-immunolocalization of RNF185 WT and RNF185 mutants with ER marker. HEK293 cells were co-transfected with the different RNF185 constructs and a plasmid expressing an ER-localized GFP. Very low doses (0.1 μg
    Figure Legend Snippet: RNF185 is an ER-localized E3 ligase. A , co-immunolocalization of RNF185 WT and RNF185 mutants with ER marker. HEK293 cells were co-transfected with the different RNF185 constructs and a plasmid expressing an ER-localized GFP. Very low doses (0.1 μg

    Techniques Used: Marker, Transfection, Construct, Plasmid Preparation, Expressing

    30) Product Images from "Cyclin-Dependent Kinase 2 Phosphorylates S/T-P Sites in the Hepadnavirus Core Protein C-Terminal Domain and Is Incorporated into Viral Capsids"

    Article Title: Cyclin-Dependent Kinase 2 Phosphorylates S/T-P Sites in the Hepadnavirus Core Protein C-Terminal Domain and Is Incorporated into Viral Capsids

    Journal: Journal of Virology

    doi: 10.1128/JVI.01218-12

    Effect of CDK inhibition on phosphorylation of GST-HCC and GST-DCC fusion proteins in HEK293T cells. HEK293T cells were transfected with plasmids to express GST-HCC141 (HBc CTD), GST-DCC196 (DHBc CTD), or GST. Three days posttransfection, the cells were labeled with [ 32 P]orthophosphate in the absence (DMSO) (A and B, lanes 1, 4, and 7) or presence of the indicated kinase inhibitors (roscovitine, 200 μM [A and B, lanes 2, 5, and 8], or CDK2 inhibitor III, 200 μM [A and B, lanes 3, 6, and 9]). GST fusion proteins were purified with GSH affinity resin. The eluted 32 P-labeled proteins were resolved by SDS-PAGE and visualized by Coomassie blue staining (A) or autoradiography (B). The fastest-migrating, presumably least-phosphorylated or nonphosphorylated, species enhanced by inhibitor treatment is indicated by the arrowhead.
    Figure Legend Snippet: Effect of CDK inhibition on phosphorylation of GST-HCC and GST-DCC fusion proteins in HEK293T cells. HEK293T cells were transfected with plasmids to express GST-HCC141 (HBc CTD), GST-DCC196 (DHBc CTD), or GST. Three days posttransfection, the cells were labeled with [ 32 P]orthophosphate in the absence (DMSO) (A and B, lanes 1, 4, and 7) or presence of the indicated kinase inhibitors (roscovitine, 200 μM [A and B, lanes 2, 5, and 8], or CDK2 inhibitor III, 200 μM [A and B, lanes 3, 6, and 9]). GST fusion proteins were purified with GSH affinity resin. The eluted 32 P-labeled proteins were resolved by SDS-PAGE and visualized by Coomassie blue staining (A) or autoradiography (B). The fastest-migrating, presumably least-phosphorylated or nonphosphorylated, species enhanced by inhibitor treatment is indicated by the arrowhead.

    Techniques Used: Inhibition, Droplet Countercurrent Chromatography, Transfection, Labeling, Purification, SDS Page, Staining, Autoradiography

    31) Product Images from "EMT Reversal in human cancer cells after IR knockdown in hyperinsulinemic mice"

    Article Title: EMT Reversal in human cancer cells after IR knockdown in hyperinsulinemic mice

    Journal: Endocrine-related cancer

    doi: 10.1530/ERC-16-0142

    Expression of IR and IGF1R in LCC6 Tumors. (A) Primary tumors from Rag/WT and Rag/MKR mice were assessed for the gene expression of the insulin receptor, demonstrating an 83% reduction of IR in the tumors from the LCC6 IRKD cells. (* p
    Figure Legend Snippet: Expression of IR and IGF1R in LCC6 Tumors. (A) Primary tumors from Rag/WT and Rag/MKR mice were assessed for the gene expression of the insulin receptor, demonstrating an 83% reduction of IR in the tumors from the LCC6 IRKD cells. (* p

    Techniques Used: Expressing, Mouse Assay

    Reduction of insulin signaling pathway in LCC6 IRKD tumors. (A) Representative blots showing protein extracted from tumor tissue and analyzed by Western blot for phospho-Akt (pAKT) and total AKT expression. B-Actin antibody used as loading control. Densitometry
    Figure Legend Snippet: Reduction of insulin signaling pathway in LCC6 IRKD tumors. (A) Representative blots showing protein extracted from tumor tissue and analyzed by Western blot for phospho-Akt (pAKT) and total AKT expression. B-Actin antibody used as loading control. Densitometry

    Techniques Used: Western Blot, Expressing

    Tumors from LCC6 IRKD cells have reversal of Epithelial-Mesenchymal Transition phenotype. (A) Representative blots showing protein extracted from tumor tissue and analyzed by Western blot for Vimentin expression. B-Actin antibody was used as the loading
    Figure Legend Snippet: Tumors from LCC6 IRKD cells have reversal of Epithelial-Mesenchymal Transition phenotype. (A) Representative blots showing protein extracted from tumor tissue and analyzed by Western blot for Vimentin expression. B-Actin antibody was used as the loading

    Techniques Used: Western Blot, Expressing

    32) Product Images from "The Plasmodium Class XIV Myosin, MyoB, Has a Distinct Subcellular Location in Invasive and Motile Stages of the Malaria Parasite and an Unusual Light Chain *"

    Article Title: The Plasmodium Class XIV Myosin, MyoB, Has a Distinct Subcellular Location in Invasive and Motile Stages of the Malaria Parasite and an Unusual Light Chain *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.637694

    Generation of PfMyoB-GFP and PfMyoA-GFP parasites. A, schematic representation of the GFP-tagging of PfMyoB by single crossover homologous recombination into the myoB locus. The primers for PCR ( arrows 1 and 2 ) and the Southern blot probe together with restriction sites are labeled. X = XbaI and H = HpaI. B, diagnostic PCR on genomic DNA showing integration of PfMyoB-GFP ( primers 3 + 5 ) and wild type ( primers 3 + 4 ). Two PfMyoBGFP clones were examined. C, Southern blot analysis of cloned PfMyoB-GFP parasites. Genomic DNA was digested with XbaI and HpaI restriction enzymes. A probe to the myob region of homology showed the following: PfMyoB-GFP cycle 0 ( c0 ) shows the presence of wild-type (8.4 kb) and episome (4.3 kb) bands; 3D7 parasites only show the wild-type band. Clone 1 shows the expected bands for integration (7.9 and 4.8 kb), but also for episome, suggesting concatamer insertion. Clone 2 shows only bands for integration and was therefore used in all subsequent experiments. D, Western blot. Extracts of late stage schizonts from 3D7 and PfMyoB-GFP clone 2 parasites were immunoblotted wth an anti-GFP antibody. MyoB-GFP protein of ∼120 kDa was detected in clone 2. E, schematic representation of the GFP tagging of MyoA by single crossover homologous recombination into the myoA locus, with primers for PCR ( arrows with primer pair 15 and 16) and Southern blot probe and restriction sites labeled. C = ClaI and B = BsrFI. F, diagnostic PCR on genomic DNA showing integration of PfMyoA-GFP ( primers 17 + 5 ) and wild type ( primers 17 + 18 ). Four PfMyoA-GFP-expressing clones were examined. G, PfMyoA-GFP-expressing merozoites as viewed by live fluorescence microscopy. GFP was detected by green fluorescence, and the nuclei ( blue ) were labeled with Hoechst dye prior to microscopic analysis; the GFP signal is distributed to the parasite periphery. Scale bar, 2 μm. H, Southern blot analysis of cloned PfMyoA-GFP-expressing parasites. Genomic DNA was digested with ClaI and BsrFI. When probed with the myoa region of homology, all clones showed the expected two integration bands at 11.1 and 2.5 kb. 3D7 is the wild-type control and shows a band of the expected size (7.3 kb).
    Figure Legend Snippet: Generation of PfMyoB-GFP and PfMyoA-GFP parasites. A, schematic representation of the GFP-tagging of PfMyoB by single crossover homologous recombination into the myoB locus. The primers for PCR ( arrows 1 and 2 ) and the Southern blot probe together with restriction sites are labeled. X = XbaI and H = HpaI. B, diagnostic PCR on genomic DNA showing integration of PfMyoB-GFP ( primers 3 + 5 ) and wild type ( primers 3 + 4 ). Two PfMyoBGFP clones were examined. C, Southern blot analysis of cloned PfMyoB-GFP parasites. Genomic DNA was digested with XbaI and HpaI restriction enzymes. A probe to the myob region of homology showed the following: PfMyoB-GFP cycle 0 ( c0 ) shows the presence of wild-type (8.4 kb) and episome (4.3 kb) bands; 3D7 parasites only show the wild-type band. Clone 1 shows the expected bands for integration (7.9 and 4.8 kb), but also for episome, suggesting concatamer insertion. Clone 2 shows only bands for integration and was therefore used in all subsequent experiments. D, Western blot. Extracts of late stage schizonts from 3D7 and PfMyoB-GFP clone 2 parasites were immunoblotted wth an anti-GFP antibody. MyoB-GFP protein of ∼120 kDa was detected in clone 2. E, schematic representation of the GFP tagging of MyoA by single crossover homologous recombination into the myoA locus, with primers for PCR ( arrows with primer pair 15 and 16) and Southern blot probe and restriction sites labeled. C = ClaI and B = BsrFI. F, diagnostic PCR on genomic DNA showing integration of PfMyoA-GFP ( primers 17 + 5 ) and wild type ( primers 17 + 18 ). Four PfMyoA-GFP-expressing clones were examined. G, PfMyoA-GFP-expressing merozoites as viewed by live fluorescence microscopy. GFP was detected by green fluorescence, and the nuclei ( blue ) were labeled with Hoechst dye prior to microscopic analysis; the GFP signal is distributed to the parasite periphery. Scale bar, 2 μm. H, Southern blot analysis of cloned PfMyoA-GFP-expressing parasites. Genomic DNA was digested with ClaI and BsrFI. When probed with the myoa region of homology, all clones showed the expected two integration bands at 11.1 and 2.5 kb. 3D7 is the wild-type control and shows a band of the expected size (7.3 kb).

    Techniques Used: Homologous Recombination, Polymerase Chain Reaction, Southern Blot, Labeling, Diagnostic Assay, Clone Assay, Western Blot, Expressing, Fluorescence, Microscopy

    PfMyoB-GFP does not associate with the glideosome components MTIP, GAP45, and GAP50. i, Western blot of parasite lysates from 3D7, MyoA-GFP ( A ), and MyoB-GFP ( B ) parasite lines. ii, GFP-TRAP immunoprecipitates from corresponding parasite lysates (shown in i ) separated by SDS-PAGE and probed with antibodies indicated on the right of each panel (rabbit anti-GFP, anti-GAP50, anti-GAP45, and anti-MTIP). Although GAP50, GAP45, and MTIP were present in all the lysates, they were detected in the MyoA-GFP immunoprecipitate but not in the MyoB-GFP immunoprecipitate. Molecular mass markers are indicated on the left in kDa.
    Figure Legend Snippet: PfMyoB-GFP does not associate with the glideosome components MTIP, GAP45, and GAP50. i, Western blot of parasite lysates from 3D7, MyoA-GFP ( A ), and MyoB-GFP ( B ) parasite lines. ii, GFP-TRAP immunoprecipitates from corresponding parasite lysates (shown in i ) separated by SDS-PAGE and probed with antibodies indicated on the right of each panel (rabbit anti-GFP, anti-GAP50, anti-GAP45, and anti-MTIP). Although GAP50, GAP45, and MTIP were present in all the lysates, they were detected in the MyoA-GFP immunoprecipitate but not in the MyoB-GFP immunoprecipitate. Molecular mass markers are indicated on the left in kDa.

    Techniques Used: Western Blot, SDS Page

    33) Product Images from "Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Non-redundant Functions in Vivo"

    Article Title: Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Non-redundant Functions in Vivo

    Journal: Current Biology

    doi: 10.1016/j.cub.2018.10.002

    The D Site in Sra-1 Is Needed for Proper Lamellipodia Formation (A) Representative Sra-1/PIR121 KO cells expressing different EGFP-Sra-1 variants, as indicated, and stained with phalloidin (scale bar, 10 μm). (B) Quantification of lamellipodial width measurements. (C) Quantification of filopodia formed anterior to lamellipodia. (D) Representative kymographs of lamellipodial protrusion, induced by the indicated EGFP-Sra-1 variants. (E) Quantification of lamellipodial protrusion velocity mediated upon expression of the respective Sra-1 variants. (F) Representative Sra-1/PIR121 KO cells expressing different EGFP-Sra-1 variants and stained for the Arp2/3 complex subunit ArpC5A (scale bar, 10 μm). (G) Quantification of ArpC5A intensity at the lamellipodium. For quantifications in (B), (C), (E), and (G), data are displayed as described for Figure 1 C, n = number of cells analyzed, and statistical significance is expressed as ∗∗∗ p ≤ 0.001 and ∗∗ p ≤ 0.01; n.s. (not significant): p > 0.05. See also Figure S3 and Video S3 .
    Figure Legend Snippet: The D Site in Sra-1 Is Needed for Proper Lamellipodia Formation (A) Representative Sra-1/PIR121 KO cells expressing different EGFP-Sra-1 variants, as indicated, and stained with phalloidin (scale bar, 10 μm). (B) Quantification of lamellipodial width measurements. (C) Quantification of filopodia formed anterior to lamellipodia. (D) Representative kymographs of lamellipodial protrusion, induced by the indicated EGFP-Sra-1 variants. (E) Quantification of lamellipodial protrusion velocity mediated upon expression of the respective Sra-1 variants. (F) Representative Sra-1/PIR121 KO cells expressing different EGFP-Sra-1 variants and stained for the Arp2/3 complex subunit ArpC5A (scale bar, 10 μm). (G) Quantification of ArpC5A intensity at the lamellipodium. For quantifications in (B), (C), (E), and (G), data are displayed as described for Figure 1 C, n = number of cells analyzed, and statistical significance is expressed as ∗∗∗ p ≤ 0.001 and ∗∗ p ≤ 0.01; n.s. (not significant): p > 0.05. See also Figure S3 and Video S3 .

    Techniques Used: Expressing, Staining

    Further D Site-Associated Phenotypes and Analysis of the D Site Interaction Surface on Rac (A and B) Determination of lamellipodial actin assembly rates. Sra-1/PIR121 KO cells (clone 3) were co-transfected with mCherry-Sra-1 variants (WT, D site mutant, and D site+WCA ∗ , as indicated) and EGFP-actin. Lamellipodial actin assembly rates were determined by photobleaching EGFP-actin within lamellipodial regions, and reading out network assembly rates as the sum of actin rearward flow during the fluorescence recovery period and simultaneous forward protrusion (A). Representative frames before and after bleaching (at 0 s) are shown in (B). Scale bar, 5 μm. (C) Quantification of F-actin intensity levels in the lamellipodium obtained from phalloidin stainings. Data and results of statistical analyses in (A) and (C) are displayed as described for quantifications in Figure 3 . (D) Rac1 docking onto the D site of Sra-1 based on the cryo-EM structure of Rac1 occupying the D site [ 3 ]. Amino acids mutated in this study (i.e., E31 and F37 in Rac1, as well as Y967 and G971 in Sra-1) are shown as sticks, except for G971, the position of which in the green helix is shown in red. Additional amino acids interacting with either E31/F37 of Rac1 or Y967 of Sra-1 are shown as lines. (E) RAC1/2/3 genes were disrupted in B16-F1 cells using CRISPR/Cas9, and derived knockout clones were assayed for Rac expression. Antibodies employed are capable of detecting Rac1 or 3 and Rac2, respectively (see [ 25 ]). All lines including B16-F1 wild-type lack expression of hematopoietic Rac2, except for RAW macrophages (MΦ) used as control, and all KO clones except for clone 2 lack full-length Rac1/3 protein. (F) Representative Rac1/2/3 KO clone (clone 1) and B16-F1 control cells plated on laminin-coated coverslips for analysis of the actin cytoskeleton by phalloidin staining. Note the complete absence of lamellipodia or lamellipodia-like structures upon elimination of Rac expression. Scale bar, 20 μm. (G) Rac1/2/3 KO clone 1 was transfected with EGFP-tagged Rac1-L61 constructs as indicated, and plated on laminin-coated coverslips for analysis of cell morphology. Representative cells are shown. Note that mutation of E31 or F37 residues in Rac1-L61 resulted in the induction of compromised lamellipodia, highly reminiscent in phenotype of those observed upon expression of D site-mutated Sra-1 in Sra-1/PIR121 double KO cells. Scale bar, 10 μm.
    Figure Legend Snippet: Further D Site-Associated Phenotypes and Analysis of the D Site Interaction Surface on Rac (A and B) Determination of lamellipodial actin assembly rates. Sra-1/PIR121 KO cells (clone 3) were co-transfected with mCherry-Sra-1 variants (WT, D site mutant, and D site+WCA ∗ , as indicated) and EGFP-actin. Lamellipodial actin assembly rates were determined by photobleaching EGFP-actin within lamellipodial regions, and reading out network assembly rates as the sum of actin rearward flow during the fluorescence recovery period and simultaneous forward protrusion (A). Representative frames before and after bleaching (at 0 s) are shown in (B). Scale bar, 5 μm. (C) Quantification of F-actin intensity levels in the lamellipodium obtained from phalloidin stainings. Data and results of statistical analyses in (A) and (C) are displayed as described for quantifications in Figure 3 . (D) Rac1 docking onto the D site of Sra-1 based on the cryo-EM structure of Rac1 occupying the D site [ 3 ]. Amino acids mutated in this study (i.e., E31 and F37 in Rac1, as well as Y967 and G971 in Sra-1) are shown as sticks, except for G971, the position of which in the green helix is shown in red. Additional amino acids interacting with either E31/F37 of Rac1 or Y967 of Sra-1 are shown as lines. (E) RAC1/2/3 genes were disrupted in B16-F1 cells using CRISPR/Cas9, and derived knockout clones were assayed for Rac expression. Antibodies employed are capable of detecting Rac1 or 3 and Rac2, respectively (see [ 25 ]). All lines including B16-F1 wild-type lack expression of hematopoietic Rac2, except for RAW macrophages (MΦ) used as control, and all KO clones except for clone 2 lack full-length Rac1/3 protein. (F) Representative Rac1/2/3 KO clone (clone 1) and B16-F1 control cells plated on laminin-coated coverslips for analysis of the actin cytoskeleton by phalloidin staining. Note the complete absence of lamellipodia or lamellipodia-like structures upon elimination of Rac expression. Scale bar, 20 μm. (G) Rac1/2/3 KO clone 1 was transfected with EGFP-tagged Rac1-L61 constructs as indicated, and plated on laminin-coated coverslips for analysis of cell morphology. Representative cells are shown. Note that mutation of E31 or F37 residues in Rac1-L61 resulted in the induction of compromised lamellipodia, highly reminiscent in phenotype of those observed upon expression of D site-mutated Sra-1 in Sra-1/PIR121 double KO cells. Scale bar, 10 μm.

    Techniques Used: Transfection, Mutagenesis, Flow Cytometry, Fluorescence, CRISPR, Derivative Assay, Knock-Out, Clone Assay, Expressing, Staining, Construct

    Contribution of Distinct Rac Binding Sites in Sra-1 to Lamellipodia Formation (A) Cell morphologies and lamellipodial phenotypes of B16-F1 control versus Sra-1/PIR121 KO cells (clone 3) transfected with EGFP or EGFP-tagged Sra-1, and stained for the actin cytoskeleton with phalloidin (scale bars, 20 μm). (B) Cell lysates of B16-F1 cells, Sra-1/PIR121 KO cells (clone 3), as well as KO cells expressing EGFP-Sra-1 were subjected to western blotting to detect expression levels of WAVE complex components, as indicated. (C) B16-F1 control cells, Sra-1/PIR121 KO clone 3, and the latter forming lamellipodia upon transfection with EGFP-tagged Sra-1 were analyzed for random migration speed ( ∗∗∗ p ≤ 0.001; n.s. [not significant]: p > 0.05). Box and whisker plots represent data as follows: boxes correspond to 50% of data points (25%–75%), and whiskers correspond to 80% (10%–90%). Outliers are shown as dots, and lines and red numbers in boxes correspond to medians. (D) Crystal structure of the WAVE complex (PDB: 3P8C [ 4 ]). From the view chosen, only WAVE (magenta), Sra-1 (green), and Nap1 (blue) are visible. Sra-1 possesses two binding sites for Rac (termed A site and D site) and sequesters the WH2 and C regions of WAVE. Rac binding to Sra-1 is thought to release interactions with the WH2 and C regions, thereby activating the WCA domain of WAVE. (E) Sra-1/PIR121 KO cells (clone 3) were transfected with EGFP or various EGFP-Sra-1 constructs, lysed, and subjected to pull-downs with constitutively active Rac1 (Rac1-L61). Note strongly increased interaction of the WCA ∗ mutant with Rac1, which was strongly and virtually entirely diminished upon additional mutation of the A and D site, respectively. Combinatorial mutation of both Rac binding sites in the WCA ∗ background appeared to abolish detectable Rac1 interaction entirely. WCA ∗ : disrupted WH2 and C region contact sites (L697D/Y704D/L841A/F844A/W845A). (F) Sra-1/PIR121 KO cells (clone 3) were transfected with the indicated EGFP-Sra-1 constructs and assayed for lamellipodia formation. Lamellipodial actin networks that were small, narrow, or displayed multiple ruffles were defined as “immature lamellipodia,” marked by arrowheads in cell images (right), as opposed to regular lamellipodia, marked by arrows (scale bar, 10 μm). Data in the bar chart are arithmetic means ± SEM from three independent experiments. Note that the A site mutation diminished lamellipodia formation in a fashion that could be restored by additional WCA ∗ mutation of Sra-1. In the case of the D site, lamellipodial morphology was compromised in a fashion mostly independent from the WCA ∗ mutation. The WIRS mutation had no detectable effect. To assess statistical significance of differences or confirm the absence of statistically relevant differences between experimental groups, a non-parametric, Mann-Whitney rank-sum test was performed in multiple, individual combinations of datasets. For each experimental group, we compared the number of cells with regular, i.e., “fully developed” lamellipodia, immature lamellipodia (see above), or the two groups combined, and hence all cells display either one of the lamellipodium-like structures. Selected combinations are as follows, with three p values representing aforementioned lamellipodial categories: WT-WIRS (n.s., n.s., n.s.); WT-C179R/R190D+WCA ∗ (n.s., n.s., n.s.); WT-Y967A ( ∗∗ , ∗∗ , n.s.); WT-G971W ( ∗∗ , ∗ , n.s.); WT-Y967A+WCA ∗ ( ∗∗ , ∗∗ , n.s.); Y967A-Y967A+WCA ∗ ( ∗ , n.s., n.s.); WT-WCA ∗ (n.s., n.s., ∗∗ ). Statistical significance is expressed as ∗∗ p ≤ 0.01, ∗ p ≤ 0.05, and n.s. (not significant): p > 0.05. WIRS: Y923A/E1084A to mutate the WIRS-binding pocket; WCA ∗ : disrupted WH2 and C region contact sites (L697D/Y704D/L841A/F844A/W845A). See also Figure S1 and Video S1 .
    Figure Legend Snippet: Contribution of Distinct Rac Binding Sites in Sra-1 to Lamellipodia Formation (A) Cell morphologies and lamellipodial phenotypes of B16-F1 control versus Sra-1/PIR121 KO cells (clone 3) transfected with EGFP or EGFP-tagged Sra-1, and stained for the actin cytoskeleton with phalloidin (scale bars, 20 μm). (B) Cell lysates of B16-F1 cells, Sra-1/PIR121 KO cells (clone 3), as well as KO cells expressing EGFP-Sra-1 were subjected to western blotting to detect expression levels of WAVE complex components, as indicated. (C) B16-F1 control cells, Sra-1/PIR121 KO clone 3, and the latter forming lamellipodia upon transfection with EGFP-tagged Sra-1 were analyzed for random migration speed ( ∗∗∗ p ≤ 0.001; n.s. [not significant]: p > 0.05). Box and whisker plots represent data as follows: boxes correspond to 50% of data points (25%–75%), and whiskers correspond to 80% (10%–90%). Outliers are shown as dots, and lines and red numbers in boxes correspond to medians. (D) Crystal structure of the WAVE complex (PDB: 3P8C [ 4 ]). From the view chosen, only WAVE (magenta), Sra-1 (green), and Nap1 (blue) are visible. Sra-1 possesses two binding sites for Rac (termed A site and D site) and sequesters the WH2 and C regions of WAVE. Rac binding to Sra-1 is thought to release interactions with the WH2 and C regions, thereby activating the WCA domain of WAVE. (E) Sra-1/PIR121 KO cells (clone 3) were transfected with EGFP or various EGFP-Sra-1 constructs, lysed, and subjected to pull-downs with constitutively active Rac1 (Rac1-L61). Note strongly increased interaction of the WCA ∗ mutant with Rac1, which was strongly and virtually entirely diminished upon additional mutation of the A and D site, respectively. Combinatorial mutation of both Rac binding sites in the WCA ∗ background appeared to abolish detectable Rac1 interaction entirely. WCA ∗ : disrupted WH2 and C region contact sites (L697D/Y704D/L841A/F844A/W845A). (F) Sra-1/PIR121 KO cells (clone 3) were transfected with the indicated EGFP-Sra-1 constructs and assayed for lamellipodia formation. Lamellipodial actin networks that were small, narrow, or displayed multiple ruffles were defined as “immature lamellipodia,” marked by arrowheads in cell images (right), as opposed to regular lamellipodia, marked by arrows (scale bar, 10 μm). Data in the bar chart are arithmetic means ± SEM from three independent experiments. Note that the A site mutation diminished lamellipodia formation in a fashion that could be restored by additional WCA ∗ mutation of Sra-1. In the case of the D site, lamellipodial morphology was compromised in a fashion mostly independent from the WCA ∗ mutation. The WIRS mutation had no detectable effect. To assess statistical significance of differences or confirm the absence of statistically relevant differences between experimental groups, a non-parametric, Mann-Whitney rank-sum test was performed in multiple, individual combinations of datasets. For each experimental group, we compared the number of cells with regular, i.e., “fully developed” lamellipodia, immature lamellipodia (see above), or the two groups combined, and hence all cells display either one of the lamellipodium-like structures. Selected combinations are as follows, with three p values representing aforementioned lamellipodial categories: WT-WIRS (n.s., n.s., n.s.); WT-C179R/R190D+WCA ∗ (n.s., n.s., n.s.); WT-Y967A ( ∗∗ , ∗∗ , n.s.); WT-G971W ( ∗∗ , ∗ , n.s.); WT-Y967A+WCA ∗ ( ∗∗ , ∗∗ , n.s.); Y967A-Y967A+WCA ∗ ( ∗ , n.s., n.s.); WT-WCA ∗ (n.s., n.s., ∗∗ ). Statistical significance is expressed as ∗∗ p ≤ 0.01, ∗ p ≤ 0.05, and n.s. (not significant): p > 0.05. WIRS: Y923A/E1084A to mutate the WIRS-binding pocket; WCA ∗ : disrupted WH2 and C region contact sites (L697D/Y704D/L841A/F844A/W845A). See also Figure S1 and Video S1 .

    Techniques Used: Binding Assay, Transfection, Staining, Expressing, Western Blot, Migration, Whisker Assay, Construct, Mutagenesis, MANN-WHITNEY

    Functional Comparison of Rac Binding Sites in Mouse and Dictyostelium Cells (A) Subcellular localization of Scar/WAVE and Arp2/3 complexes. Wild-type and mutated Pir121-EGFP were expressed in PirA knockouts and visualized by confocal microscopy while migrating under agarose up a folate gradient. Top: wild-type Pir121-EGFP; middle: A site mutant (K193D/R194D); bottom: D site mutant (Y961A). Arp2/3 complex (red) is recruited to sites of WT Scar/WAVE complex (green) localization, but not upon expression of either PIR121 mutant. The D site mutant allows its recruitment to pseudopods, but there is no detectable Arp2/3 complex activation. (B) Lifetimes of Scar/WAVE patches. Cells expressing PIR121-EGFP were allowed to migrate up folate gradients under agarose, and areas of local EGFP enrichment were observed by confocal microscopy. A site mutants showed no patches. Graph shows means ± SD; n > 25 cells; ∗∗∗ p ≤ 0.001, non-parametric t test, Mann-Whitney. (C) Frequency of Scar/WAVE patch generation. Cells were measured as in (B), but the rate of formation of patches was quantitated. Graph shows means ± SD; n > 25 cells; ∗∗∗ p ≤ 0.001, non-parametric t test, Mann-Whitney. (D) Reduced migration speed of cells expressing both A and D site mutants. The chemotactic speeds of cells expressing WT and mutant PIR121 were measured from the speed of cells allowed to migrate up folate gradients under agarose, and observed by differential interference contrast (DIC) microscopy. Data show means ± SD; n > 25 cells on 3 days; ∗∗∗ p ≤ 0.001, one-way ANOVA, Dunnett’s multiple-comparison test. (E) Random migration assay with B16-F1 Sra-1/PIR121 KO cells (clone 3) re-expressing the indicated Sra-1 variants, and analyzed as described in STAR Methods ; cells with and without lamellipodia are displayed separately. n of cells analyzed was ≥130 per condition and specifically indicated for cells harboring lamellipodia. Lamellipodia-forming cells always migrated faster than respective controls, i.e., those cells lacking lamellipodia in each condition. Statistical significance is expressed as ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.01, and ∗ p ≤ 0.05; n.s. (not significant): p > 0.05. (F and G) FRAP analysis of EGFP-Sra-1 variants expressed in Sra-1/PIR121 KO B16-F1 cells (clone 3). Data are arithmetic means with SEM of fluorescence intensities at acquired time points after bleaching, with intensities before each bleach individually normalized to 1. “n” equals the number of individual FRAP videos analyzed for each component (F). Half-times of recovery for each component were derived from curve fits (not shown) generated as described in STAR Methods . In (G), fitted data curves are displayed for comparison upon normalization of bleaching time point (0 s) to 0 and fluorescence recovery asymptote to 1. Constructs used were: WCA ∗ : disrupted WH2 and C region contact sites (L697D/Y704D/L841A/F844A/W845A); A site: C179R/R190D; D site: Y967A. See also Figures S2 and S3 and Videos S2 and S4 .
    Figure Legend Snippet: Functional Comparison of Rac Binding Sites in Mouse and Dictyostelium Cells (A) Subcellular localization of Scar/WAVE and Arp2/3 complexes. Wild-type and mutated Pir121-EGFP were expressed in PirA knockouts and visualized by confocal microscopy while migrating under agarose up a folate gradient. Top: wild-type Pir121-EGFP; middle: A site mutant (K193D/R194D); bottom: D site mutant (Y961A). Arp2/3 complex (red) is recruited to sites of WT Scar/WAVE complex (green) localization, but not upon expression of either PIR121 mutant. The D site mutant allows its recruitment to pseudopods, but there is no detectable Arp2/3 complex activation. (B) Lifetimes of Scar/WAVE patches. Cells expressing PIR121-EGFP were allowed to migrate up folate gradients under agarose, and areas of local EGFP enrichment were observed by confocal microscopy. A site mutants showed no patches. Graph shows means ± SD; n > 25 cells; ∗∗∗ p ≤ 0.001, non-parametric t test, Mann-Whitney. (C) Frequency of Scar/WAVE patch generation. Cells were measured as in (B), but the rate of formation of patches was quantitated. Graph shows means ± SD; n > 25 cells; ∗∗∗ p ≤ 0.001, non-parametric t test, Mann-Whitney. (D) Reduced migration speed of cells expressing both A and D site mutants. The chemotactic speeds of cells expressing WT and mutant PIR121 were measured from the speed of cells allowed to migrate up folate gradients under agarose, and observed by differential interference contrast (DIC) microscopy. Data show means ± SD; n > 25 cells on 3 days; ∗∗∗ p ≤ 0.001, one-way ANOVA, Dunnett’s multiple-comparison test. (E) Random migration assay with B16-F1 Sra-1/PIR121 KO cells (clone 3) re-expressing the indicated Sra-1 variants, and analyzed as described in STAR Methods ; cells with and without lamellipodia are displayed separately. n of cells analyzed was ≥130 per condition and specifically indicated for cells harboring lamellipodia. Lamellipodia-forming cells always migrated faster than respective controls, i.e., those cells lacking lamellipodia in each condition. Statistical significance is expressed as ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.01, and ∗ p ≤ 0.05; n.s. (not significant): p > 0.05. (F and G) FRAP analysis of EGFP-Sra-1 variants expressed in Sra-1/PIR121 KO B16-F1 cells (clone 3). Data are arithmetic means with SEM of fluorescence intensities at acquired time points after bleaching, with intensities before each bleach individually normalized to 1. “n” equals the number of individual FRAP videos analyzed for each component (F). Half-times of recovery for each component were derived from curve fits (not shown) generated as described in STAR Methods . In (G), fitted data curves are displayed for comparison upon normalization of bleaching time point (0 s) to 0 and fluorescence recovery asymptote to 1. Constructs used were: WCA ∗ : disrupted WH2 and C region contact sites (L697D/Y704D/L841A/F844A/W845A); A site: C179R/R190D; D site: Y967A. See also Figures S2 and S3 and Videos S2 and S4 .

    Techniques Used: Functional Assay, Binding Assay, Confocal Microscopy, Mutagenesis, Expressing, Activation Assay, MANN-WHITNEY, Migration, Microscopy, Fluorescence, Derivative Assay, Generated, Construct

    34) Product Images from "NSs Protein of Rift Valley Fever Virus Promotes Posttranslational Downregulation of the TFIIH Subunit p62 ▿"

    Article Title: NSs Protein of Rift Valley Fever Virus Promotes Posttranslational Downregulation of the TFIIH Subunit p62 ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02255-10

    The proteasome is involved in p62 downregulation. (A) 293 cells were pretreated with 50 μM lactacystin (lac) or 5 μM MG132 for 30 min to inhibit proteasomal degradation before being mock transfected or transfected with in vitro -synthesized
    Figure Legend Snippet: The proteasome is involved in p62 downregulation. (A) 293 cells were pretreated with 50 μM lactacystin (lac) or 5 μM MG132 for 30 min to inhibit proteasomal degradation before being mock transfected or transfected with in vitro -synthesized

    Techniques Used: Transfection, In Vitro, Synthesized

    35) Product Images from "Galectin-9 binds IgM-BCR to regulate B cell signaling"

    Article Title: Galectin-9 binds IgM-BCR to regulate B cell signaling

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05771-8

    Schematic model of galectin-9 regulation of B-cell activation. a In resting primary naive WT B cells, galectin-9 facilitates interactions between BCRs and either the inhibitory proteins CD45 or CD22 through binding to N-linked glycans, providing a basal attenuation of B-cell signaling upon antigen stimulation. b BCR signaling is enhanced in Gal9-KO B cells due to loss of association of BCR with inhibitory co-receptors. c Treatment of WT B cells with rGal9 induces the association of IgM-BCR with CD45 and CD22 to suppress B-cell signaling
    Figure Legend Snippet: Schematic model of galectin-9 regulation of B-cell activation. a In resting primary naive WT B cells, galectin-9 facilitates interactions between BCRs and either the inhibitory proteins CD45 or CD22 through binding to N-linked glycans, providing a basal attenuation of B-cell signaling upon antigen stimulation. b BCR signaling is enhanced in Gal9-KO B cells due to loss of association of BCR with inhibitory co-receptors. c Treatment of WT B cells with rGal9 induces the association of IgM-BCR with CD45 and CD22 to suppress B-cell signaling

    Techniques Used: Activation Assay, Binding Assay

    Galectin-9 alters IgM-BCR nanoclusters. a TIRFM image of surface IgM and fluorescently labeled rGal9 before bleaching for image acquisition (two left panels respectively). dSTORM images reconstructed from single-molecule localization processed by Thunderstorm software mapped to a fire color scale as indicated; the magnified region (3 × 3 µm) from ROI (white box) is shown as 2D image (middle) and 3D surface plot (right) in the order of WT (top), Gal9-KO (middle), and Gal9-KO + 1 µM rGal9 (bottom). Scale bar represents 2 µm. b Quantification of the distribution of IgM by H function and c Hopkins index of localizations inside ROIs. d – g Reconstructed images were analyzed by a model-based Bayesian approach to identify nanoclusters and their physical properties. d Number of clusters (one point per ROI). e Cluster radii (one point per cluster). f Number of molecules (one point per cluster). g Percentage of localization in clusters (one point per ROI). Each category contains at least 15 ROIs from three independent experiments (at least four cells per experiment). Statistical analysis was performed using Kruskal-Wallis test with Dunn’s multiple comparison test ( d , e , f ) and one-way ANOVA with Tukey’s multiple comparison test ( b , g ). Red bars indicate mean ± SEM; * p
    Figure Legend Snippet: Galectin-9 alters IgM-BCR nanoclusters. a TIRFM image of surface IgM and fluorescently labeled rGal9 before bleaching for image acquisition (two left panels respectively). dSTORM images reconstructed from single-molecule localization processed by Thunderstorm software mapped to a fire color scale as indicated; the magnified region (3 × 3 µm) from ROI (white box) is shown as 2D image (middle) and 3D surface plot (right) in the order of WT (top), Gal9-KO (middle), and Gal9-KO + 1 µM rGal9 (bottom). Scale bar represents 2 µm. b Quantification of the distribution of IgM by H function and c Hopkins index of localizations inside ROIs. d – g Reconstructed images were analyzed by a model-based Bayesian approach to identify nanoclusters and their physical properties. d Number of clusters (one point per ROI). e Cluster radii (one point per cluster). f Number of molecules (one point per cluster). g Percentage of localization in clusters (one point per ROI). Each category contains at least 15 ROIs from three independent experiments (at least four cells per experiment). Statistical analysis was performed using Kruskal-Wallis test with Dunn’s multiple comparison test ( d , e , f ) and one-way ANOVA with Tukey’s multiple comparison test ( b , g ). Red bars indicate mean ± SEM; * p

    Techniques Used: Labeling, Software

    Galectin-9 increases colocalization between CD22 and IgM in primary B cells. a Representative merged TIRF (top), dSTORM (middle), and dSTORM zoom (bottom) images showing surface CD45 (magenta) and IgM-BCR (green) on primary wild-type (WT) (left) and galectin-9 knockout (Gal9-KO) (right) B cells. dSTORM ROI (3 × 3 μm) is outlined in yellow (middle) and magnified in dSTORM zoom (bottom). b – e Quantification of at least 20 ROIs from WT and Gal9-KO B cells pooled from three independent experiments. b Hopkin’s index showing randomness of CD45 organization (one point per ROI). c H function derived from Riplely’s K showing degree of CD45 clustering. d Mean diameter of CD45 clusters (one point per ROI). e Mean area of CD45 clusters (one point per ROI). f , g Quantification of at least 15 ROIs from WT and Gal9-KO B cells pooled from three independent experiments. f Coordinate-based colocalization (CBC) histograms of the single-molecule distributions of colocalizations between CD45 and IgM. g Nearest-neighbor distance (NND) analysis of the data shown in f . Symbol represents the median NND of all paired single-molecule localizations from one ROI. h Representative merged TIRF and dSTORM images showing surface CD22 (magenta) and IgM-BCR (green) on primary WT (left) and Gal9-KO (right) B cells. i – l Quantification of at least 30 ROIs from WT and Gal9-KO B cells pooled from three independent experiments. i Mean Hopkin’s index showing randomness of CD22 organization (one point per ROI). j H function showing degree of CD22 clustering. k Mean diameter of CD22 clusters (one point per ROI). l Mean area of CD22 clusters (one point per ROI). m , n Quantification of at least 20 ROIs from WT and Gal9-KO B cells pooled from three independent experiments. m CBC histograms of the single-molecule distributions of colocalizations between CD22 and IgM. n NND analysis of the data shown in m . Colocalization between channels shown in white. Scale bars represent 2 and 1 μm (zoom). Mean ± SEM indicated by the red bar. Statistical significance assessed by Mann-Whitney, * p
    Figure Legend Snippet: Galectin-9 increases colocalization between CD22 and IgM in primary B cells. a Representative merged TIRF (top), dSTORM (middle), and dSTORM zoom (bottom) images showing surface CD45 (magenta) and IgM-BCR (green) on primary wild-type (WT) (left) and galectin-9 knockout (Gal9-KO) (right) B cells. dSTORM ROI (3 × 3 μm) is outlined in yellow (middle) and magnified in dSTORM zoom (bottom). b – e Quantification of at least 20 ROIs from WT and Gal9-KO B cells pooled from three independent experiments. b Hopkin’s index showing randomness of CD45 organization (one point per ROI). c H function derived from Riplely’s K showing degree of CD45 clustering. d Mean diameter of CD45 clusters (one point per ROI). e Mean area of CD45 clusters (one point per ROI). f , g Quantification of at least 15 ROIs from WT and Gal9-KO B cells pooled from three independent experiments. f Coordinate-based colocalization (CBC) histograms of the single-molecule distributions of colocalizations between CD45 and IgM. g Nearest-neighbor distance (NND) analysis of the data shown in f . Symbol represents the median NND of all paired single-molecule localizations from one ROI. h Representative merged TIRF and dSTORM images showing surface CD22 (magenta) and IgM-BCR (green) on primary WT (left) and Gal9-KO (right) B cells. i – l Quantification of at least 30 ROIs from WT and Gal9-KO B cells pooled from three independent experiments. i Mean Hopkin’s index showing randomness of CD22 organization (one point per ROI). j H function showing degree of CD22 clustering. k Mean diameter of CD22 clusters (one point per ROI). l Mean area of CD22 clusters (one point per ROI). m , n Quantification of at least 20 ROIs from WT and Gal9-KO B cells pooled from three independent experiments. m CBC histograms of the single-molecule distributions of colocalizations between CD22 and IgM. n NND analysis of the data shown in m . Colocalization between channels shown in white. Scale bars represent 2 and 1 μm (zoom). Mean ± SEM indicated by the red bar. Statistical significance assessed by Mann-Whitney, * p

    Techniques Used: Knock-Out, Derivative Assay, MANN-WHITNEY

    rGal9 induces coalescence of lipid raft domains containing CD22 and CD45. a Representative confocal images of primary WT B cells treated with 1 μM rGal9 and immunostained for CD45 (cyan) and galectin-9 (Gal9; yellow), and fluorescent cholera toxin (CT-B; gray) to label lipid rafts. b Fluorescence intensity profile of CD45, CT-B, and Gal9 along the cell membrane. c Representative example of masking output of algorithm to detect regions of high CT-B (lipid raft high; LR high ) and low CT-B (LR low ). d Mean fluorescence intensity of CD45 (left) and Gal9 (right) in LR low and LR high regions. e Representative confocal images of WT B cells treated with 1 μM rGal9 and immunostained for CD22 (cyan), Gal9 (yellow), and fluorescent CT-B (gray). f Fluorescence intensity profile of CD22, CT-B, and Gal9 along the cell membrane. g Mean fluorescence intensity of CD22 (left) and Gal9 (right) in LR low and LR high regions. Data are representative of at least three independent experiments. Each dot represents 1 cell, at least 30 cells measured per condition per experiment. Mean ± SEM indicated by the red bar. Statistical significance assessed by Mann-Whitney; **** p
    Figure Legend Snippet: rGal9 induces coalescence of lipid raft domains containing CD22 and CD45. a Representative confocal images of primary WT B cells treated with 1 μM rGal9 and immunostained for CD45 (cyan) and galectin-9 (Gal9; yellow), and fluorescent cholera toxin (CT-B; gray) to label lipid rafts. b Fluorescence intensity profile of CD45, CT-B, and Gal9 along the cell membrane. c Representative example of masking output of algorithm to detect regions of high CT-B (lipid raft high; LR high ) and low CT-B (LR low ). d Mean fluorescence intensity of CD45 (left) and Gal9 (right) in LR low and LR high regions. e Representative confocal images of WT B cells treated with 1 μM rGal9 and immunostained for CD22 (cyan), Gal9 (yellow), and fluorescent CT-B (gray). f Fluorescence intensity profile of CD22, CT-B, and Gal9 along the cell membrane. g Mean fluorescence intensity of CD22 (left) and Gal9 (right) in LR low and LR high regions. Data are representative of at least three independent experiments. Each dot represents 1 cell, at least 30 cells measured per condition per experiment. Mean ± SEM indicated by the red bar. Statistical significance assessed by Mann-Whitney; **** p

    Techniques Used: Fluorescence, MANN-WHITNEY

    Galectin-9 immobilizes IgM-BCR and attenuates BCR microclustering. a Diffusion coefficients and b frequency distribution histogram of single-particle tracking of IgM in WT (black circle) or Gal9-KO (blue diamond) primary B cells with the median indicated in red. Five hundred representative diffusion coefficients from a total of at least 1500 tracks from three independent experiments. c Representative TIRF image of fluorescently labeled rGal9 on primary B cell (left) and mask (right) created to differentiate tracks inside Gal9 regions (lower left, red lines) and tracks outside Gal9 regions (lower right, yellow lines). d Diffusion coefficients and e frequency distribution inside Gal9 regions (black circle) and outside Gal9 regions (black triangle) with the median indicated in red. In all, 250 representative diffusion coefficients from a total of at least 900 tracks from three independent experiments. f Representative TIRF microscopy images of WT cell (top) and WT cells treated with 1 μM rGal9 (bottom) on artificial planar lipid bilayers containing anti-kappa mapped to an 8-bit fire color scale (ImageJ). g Quantification of the total antigen intensity at the cell-bilayer interface in WT (black circles) and WT treated with rGal9 (open circles), with the mean ± SEM indicated by the red bar. Scale bar represents 2 μm. Statistical significance assessed by Mann-Whitney; **** p
    Figure Legend Snippet: Galectin-9 immobilizes IgM-BCR and attenuates BCR microclustering. a Diffusion coefficients and b frequency distribution histogram of single-particle tracking of IgM in WT (black circle) or Gal9-KO (blue diamond) primary B cells with the median indicated in red. Five hundred representative diffusion coefficients from a total of at least 1500 tracks from three independent experiments. c Representative TIRF image of fluorescently labeled rGal9 on primary B cell (left) and mask (right) created to differentiate tracks inside Gal9 regions (lower left, red lines) and tracks outside Gal9 regions (lower right, yellow lines). d Diffusion coefficients and e frequency distribution inside Gal9 regions (black circle) and outside Gal9 regions (black triangle) with the median indicated in red. In all, 250 representative diffusion coefficients from a total of at least 900 tracks from three independent experiments. f Representative TIRF microscopy images of WT cell (top) and WT cells treated with 1 μM rGal9 (bottom) on artificial planar lipid bilayers containing anti-kappa mapped to an 8-bit fire color scale (ImageJ). g Quantification of the total antigen intensity at the cell-bilayer interface in WT (black circles) and WT treated with rGal9 (open circles), with the mean ± SEM indicated by the red bar. Scale bar represents 2 μm. Statistical significance assessed by Mann-Whitney; **** p

    Techniques Used: Diffusion-based Assay, Single-particle Tracking, Labeling, Microscopy, MANN-WHITNEY

    The galectin-9 lattice increases the molecular density of IgM-BCR and co-receptors. a Representative confocal images of primary WT B cells treated with 1 µM rGal9 and immuostained for CD45 (cyan), IgM (magenta), and galectin-9 (Gal9; yellow). b Fluorescence intensity profile of CD45, IgM, and Gal9 along the cell membrane. c Representative example of masking output of algorithm to detect regions of high galectin-9 (Gal9 high ) and low galectin-9 (Gal9 low ). d Mean fluorescence intensity of CD45 (left) and IgM (right) in Gal9 high and Gal9 low regions. e Representative confocal images of WT B cells treated with 1 µM rGal9 and immunostained for CD22 (cyan), IgM (magenta), and Gal9 (yellow). f Fluorescence intensity profile of CD22, IgM, and Gal9 along the cell membrane. g Mean fluorescence intensity of CD22 (left) and IgM (right) in Gal9 high and Gal9 low regions. h Representative confocal images of WT B cells treated with 1 μM rGal9 and immunostained for CD19, IgM, and Gal9. i Fluorescence intensity profile of CD19, IgM, and Gal9 along the cell membrane. j Mean fluorescence intensity of CD19 (left) and IgM (right) in Gal9 low and Gal9 high regions. Data representative of at least three independent experiments. Each dot represents 1 cell, at least 30 cells measured per condition per experiment. Mean ± SEM indicated by the red bar. Statistical significance assessed by Mann-Whitney; **** p
    Figure Legend Snippet: The galectin-9 lattice increases the molecular density of IgM-BCR and co-receptors. a Representative confocal images of primary WT B cells treated with 1 µM rGal9 and immuostained for CD45 (cyan), IgM (magenta), and galectin-9 (Gal9; yellow). b Fluorescence intensity profile of CD45, IgM, and Gal9 along the cell membrane. c Representative example of masking output of algorithm to detect regions of high galectin-9 (Gal9 high ) and low galectin-9 (Gal9 low ). d Mean fluorescence intensity of CD45 (left) and IgM (right) in Gal9 high and Gal9 low regions. e Representative confocal images of WT B cells treated with 1 µM rGal9 and immunostained for CD22 (cyan), IgM (magenta), and Gal9 (yellow). f Fluorescence intensity profile of CD22, IgM, and Gal9 along the cell membrane. g Mean fluorescence intensity of CD22 (left) and IgM (right) in Gal9 high and Gal9 low regions. h Representative confocal images of WT B cells treated with 1 μM rGal9 and immunostained for CD19, IgM, and Gal9. i Fluorescence intensity profile of CD19, IgM, and Gal9 along the cell membrane. j Mean fluorescence intensity of CD19 (left) and IgM (right) in Gal9 low and Gal9 high regions. Data representative of at least three independent experiments. Each dot represents 1 cell, at least 30 cells measured per condition per experiment. Mean ± SEM indicated by the red bar. Statistical significance assessed by Mann-Whitney; **** p

    Techniques Used: Fluorescence, MANN-WHITNEY

    Galectin-9 is bound to the surface of primary naive B cells. a Representative flow cytometry plot (left) and quantification (right) of geometric mean ± SEM of surface staining for galectin-9 in WT (black) and Gal9-KO (blue) B cells from nine independent experiments. b Representative DIC (left) and confocal microscopy images (right) mapped to an 8-bit fire color scale (ImageJ) of primary WT (top) and Gal9-KO B cells (bottom) stained for surface galectin-9. Quantification of number of galectin-9 puncta is shown on the right (each dot represents 1 cell, 20 cells measured per condition) with the mean ± SEM indicated by the red bar. Scale bar 2 μm. Data representative of three independent experiments. c Representative confocal microscopy images of cryosections of the inguinal lymph node of WT B cells stained for subcapsular sinus macrophages (CD169; blue), B cells (B220; magenta), and Gal9 (green). Scale bar 20 μm. Data representative of three independent experiments. Statistical significance was assessed by Mann-Whitney, **** p
    Figure Legend Snippet: Galectin-9 is bound to the surface of primary naive B cells. a Representative flow cytometry plot (left) and quantification (right) of geometric mean ± SEM of surface staining for galectin-9 in WT (black) and Gal9-KO (blue) B cells from nine independent experiments. b Representative DIC (left) and confocal microscopy images (right) mapped to an 8-bit fire color scale (ImageJ) of primary WT (top) and Gal9-KO B cells (bottom) stained for surface galectin-9. Quantification of number of galectin-9 puncta is shown on the right (each dot represents 1 cell, 20 cells measured per condition) with the mean ± SEM indicated by the red bar. Scale bar 2 μm. Data representative of three independent experiments. c Representative confocal microscopy images of cryosections of the inguinal lymph node of WT B cells stained for subcapsular sinus macrophages (CD169; blue), B cells (B220; magenta), and Gal9 (green). Scale bar 20 μm. Data representative of three independent experiments. Statistical significance was assessed by Mann-Whitney, **** p

    Techniques Used: Flow Cytometry, Cytometry, Staining, Confocal Microscopy, MANN-WHITNEY

    Galectin-9 regulates BCR microcluster formation and signaling. a Representative images of primary naive WT (top) and Gal9-KO B cells (bottom) fixed on bilayers containing anti-kappa as surrogate antigen (Ag) after 90 s of spreading and imaged by confocal microscopy. Brightfield (left) and confocal (right) visualizing antigen mapped to an 8-bit fire color scale (ImageJ). Scale bar 2 μm. Quantification of b area of spreading, c total antigen fluorescence intensity at the cell-bilayer contact, and d mean intensity of antigen for WT (black circles) and Gal9-KO (blue diamonds) cells (each dot represents 1 cell, 200 cells measured per condition), with the mean ± SEM indicated by the red bar, **** p
    Figure Legend Snippet: Galectin-9 regulates BCR microcluster formation and signaling. a Representative images of primary naive WT (top) and Gal9-KO B cells (bottom) fixed on bilayers containing anti-kappa as surrogate antigen (Ag) after 90 s of spreading and imaged by confocal microscopy. Brightfield (left) and confocal (right) visualizing antigen mapped to an 8-bit fire color scale (ImageJ). Scale bar 2 μm. Quantification of b area of spreading, c total antigen fluorescence intensity at the cell-bilayer contact, and d mean intensity of antigen for WT (black circles) and Gal9-KO (blue diamonds) cells (each dot represents 1 cell, 200 cells measured per condition), with the mean ± SEM indicated by the red bar, **** p

    Techniques Used: Confocal Microscopy, Fluorescence

    Treatment with exogenous galectin-9 suppresses BCR signaling. a Representative flow cytometric histograms of WT (left) and Gal9-KO (middle) B cells either untreated or treated with various concentrations of recombinant galectin-9 (rGal9; 0.1, 0.2, 0.5, and 1 μM) followed by surface staining for galectin-9 and analyzed using flow cytometry. Overlay of endogenous galectin-9 surface expression in WT cells, and Gal9-KO cells treated with 0.1 μM rGal9 (right). b Naive B cells from WT and Gal9-KO mice treated with 0.1 μM rGal9 were settled onto anti-IgM-coated plates for the indicated time. Cells were lysed and subjected to SDS-PAGE followed by immunoblotting with anti-phospho ERK1/2 and anti-β tubulin (left panel). Quantification of the fold change in pERK over time, averaged over two independent experiments with the mean ± SEM indicated by the bar (right panel). c – f Naive B cells from WT mice were treated with 1 μM rGal9 and settled onto anti-IgM-coated plates for the indicated time. Cells were lysed and subjected to SDS-PAGE followed by immunoblotting with c anti-phosphotyrosine and ERK1/2, d anti-phospho-CD19, e anti-phospho-Akt, and f anti-phospho ERK1/2 and anti-β tubulin. Quantification of the fold change in pCD19, pAkt, and pERK over time, averaged over three independent experiments, with the mean ± SEM indicated by the bar is shown in the right panel. Statistical significance measure by two-way ANOVA followed by Sidak’s multiple comparisons test; **** p
    Figure Legend Snippet: Treatment with exogenous galectin-9 suppresses BCR signaling. a Representative flow cytometric histograms of WT (left) and Gal9-KO (middle) B cells either untreated or treated with various concentrations of recombinant galectin-9 (rGal9; 0.1, 0.2, 0.5, and 1 μM) followed by surface staining for galectin-9 and analyzed using flow cytometry. Overlay of endogenous galectin-9 surface expression in WT cells, and Gal9-KO cells treated with 0.1 μM rGal9 (right). b Naive B cells from WT and Gal9-KO mice treated with 0.1 μM rGal9 were settled onto anti-IgM-coated plates for the indicated time. Cells were lysed and subjected to SDS-PAGE followed by immunoblotting with anti-phospho ERK1/2 and anti-β tubulin (left panel). Quantification of the fold change in pERK over time, averaged over two independent experiments with the mean ± SEM indicated by the bar (right panel). c – f Naive B cells from WT mice were treated with 1 μM rGal9 and settled onto anti-IgM-coated plates for the indicated time. Cells were lysed and subjected to SDS-PAGE followed by immunoblotting with c anti-phosphotyrosine and ERK1/2, d anti-phospho-CD19, e anti-phospho-Akt, and f anti-phospho ERK1/2 and anti-β tubulin. Quantification of the fold change in pCD19, pAkt, and pERK over time, averaged over three independent experiments, with the mean ± SEM indicated by the bar is shown in the right panel. Statistical significance measure by two-way ANOVA followed by Sidak’s multiple comparisons test; **** p

    Techniques Used: Flow Cytometry, Recombinant, Staining, Cytometry, Expressing, Mouse Assay, SDS Page

    36) Product Images from "The E3 Ubiquitin Ligase TRIM21 Promotes HBV DNA Polymerase Degradation"

    Article Title: The E3 Ubiquitin Ligase TRIM21 Promotes HBV DNA Polymerase Degradation

    Journal: Viruses

    doi: 10.3390/v12030346

    TRIM21 mediates the K48-linked ubiquitination of HBV DNA Pol at K260 and K283. ( A ) A schematic diagram of the lysine point mutations of ubiquitin. ( B ) Overexpression of HBV DNA Pol and TRIM21 in Huh7 cells cotransfected with HA-ubiquitin, Ub-K48R or Ub-K63R mutants. A ubiquitination assay and Western blot were used to detect the ubiquitination and protein level of HBV DNA Pol. ( C ) Schematic of the potential ubiquitination sites of HBV DNA Pol. ( D ) Huh7 cells were transfected with FLAG-HBV DNA Pol or its lysine point mutants, and ubiquitination assays were used to analyze the ubiquitination level of HBV DNA Pol and its lysine mutants. ( E ) TRIM21 and FLAG-HBV DNA Pol or its lysine mutants were cotransfected into Huh7 cells, and the effect of TRIM21 on the ubiquitination level of HBV DNA Pol and its lysine mutants was detected by ubiquitination assay. Data are representative of three independent experiments with three replicates each.
    Figure Legend Snippet: TRIM21 mediates the K48-linked ubiquitination of HBV DNA Pol at K260 and K283. ( A ) A schematic diagram of the lysine point mutations of ubiquitin. ( B ) Overexpression of HBV DNA Pol and TRIM21 in Huh7 cells cotransfected with HA-ubiquitin, Ub-K48R or Ub-K63R mutants. A ubiquitination assay and Western blot were used to detect the ubiquitination and protein level of HBV DNA Pol. ( C ) Schematic of the potential ubiquitination sites of HBV DNA Pol. ( D ) Huh7 cells were transfected with FLAG-HBV DNA Pol or its lysine point mutants, and ubiquitination assays were used to analyze the ubiquitination level of HBV DNA Pol and its lysine mutants. ( E ) TRIM21 and FLAG-HBV DNA Pol or its lysine mutants were cotransfected into Huh7 cells, and the effect of TRIM21 on the ubiquitination level of HBV DNA Pol and its lysine mutants was detected by ubiquitination assay. Data are representative of three independent experiments with three replicates each.

    Techniques Used: Over Expression, Ubiquitin Assay, Western Blot, Transfection

    TRIM21 negatively regulates the stability of HBV DNA Pol. ( A ) Huh7 cells were transfected with TRIM21 overexpression or knockdown plasmids or control, and the level of TRIM21 mRNA or protein in the cells was detected by RT-qPCR and Western blot. ( B ) FLAG-HBV DNA Pol was cotransfected with TRIM21 overexpression or knockdown plasmid. The mRNA level of HBV DNA Pol was detected by RT-qPCR. ( C ) Huh7 cells were transfected as described in ( B ), and HepG2.2.15 cells were transfected with TRIM21 overexpression or knockdown plasmids. Western blot was used to detect the protein level of HBV DNA Pol with anti-FLAG antibody or anti-Pol antibody. ( D ) Huh7 cells were transfected with FLAG-HBV DNA Pol along with increasing amounts of TRIM21 expressing plasmid, and WWestern blot was used to detect the protein level of HBV DNA Pol. ( E ) As described in ( C ), except for using pshR-TRIM21 plasmid. ( F ) As described in ( C ), except for using TRIM21-ΔRING plasmid. ( G ) As described in ( C ), except for using TRIM21-ΔSPRY plasmid. ( H ) Huh7 cells were cotransfected with FLAG-HBV DNA Pol and pcDNA3 or TRIM21 plasmid. Thirty hours later, 100μg/ml cycloheximide was used to treat cells, and the protein level of HBV DNA Pol was detected by Western blot. ( I ) Huh7 cells were cotransfected with FLAG-HBV DNA Pol and pSilencer NC or pshR-TRIM21. The treatment was the same as in ( H ). Data are representative of three independent experiments with three replicates each.
    Figure Legend Snippet: TRIM21 negatively regulates the stability of HBV DNA Pol. ( A ) Huh7 cells were transfected with TRIM21 overexpression or knockdown plasmids or control, and the level of TRIM21 mRNA or protein in the cells was detected by RT-qPCR and Western blot. ( B ) FLAG-HBV DNA Pol was cotransfected with TRIM21 overexpression or knockdown plasmid. The mRNA level of HBV DNA Pol was detected by RT-qPCR. ( C ) Huh7 cells were transfected as described in ( B ), and HepG2.2.15 cells were transfected with TRIM21 overexpression or knockdown plasmids. Western blot was used to detect the protein level of HBV DNA Pol with anti-FLAG antibody or anti-Pol antibody. ( D ) Huh7 cells were transfected with FLAG-HBV DNA Pol along with increasing amounts of TRIM21 expressing plasmid, and WWestern blot was used to detect the protein level of HBV DNA Pol. ( E ) As described in ( C ), except for using pshR-TRIM21 plasmid. ( F ) As described in ( C ), except for using TRIM21-ΔRING plasmid. ( G ) As described in ( C ), except for using TRIM21-ΔSPRY plasmid. ( H ) Huh7 cells were cotransfected with FLAG-HBV DNA Pol and pcDNA3 or TRIM21 plasmid. Thirty hours later, 100μg/ml cycloheximide was used to treat cells, and the protein level of HBV DNA Pol was detected by Western blot. ( I ) Huh7 cells were cotransfected with FLAG-HBV DNA Pol and pSilencer NC or pshR-TRIM21. The treatment was the same as in ( H ). Data are representative of three independent experiments with three replicates each.

    Techniques Used: Transfection, Over Expression, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Expressing

    TRIM21 interacts with HBV DNA Pol. ( A ) Huh7 cells were transfected with FLAG-HBV DNA Pol and vector control, and FLAG affinity beads were used to precipitate all proteins that might interact with HBV DNA Pol. The interacting proteins of HBV DNA Pol were screened by mass spectrometry. Some potential interacting partners of HBV DNA Pol are listed. Red marks the target protein, blue marks proteins that have been reported to interact with HBV DNA Pol. Data are representative of two independent experiments. ( B ) The FLAG-HBV DNA Pol expression plasmid was transfected into Huh7 cells. After 36h, a co-IP assay was carried out with anti-FLAG antibody and control IgG antibody. HBV DNA Pol was then detected with anti-FLAG antibody, and anti-TRIM21 antibody was used to detect the endogenous expression of TRIM21 in Huh7 cells by Western blot. ( C ) Huh7 cells were cotransfected with FLAG-HBV DNA Pol and HA-TRIM21, and co-IP was performed as described in ( B ) to detect the expression of HBV DNA Pol and TRIM21. ( D ) Huh7 cells were transfected as described in ( C ), co-IP was performed with anti-HA antibody, and HBV DNA Pol and HA-TRIM21 expression were detected by Western blot. ( E ) GST-HBV DNA Pol and HA-TRIM21 were transfected into Huh7 cells, 36h after transfection, GST beads were used to pull down GST-Pol with its interacting proteins. HBV DNA Pol was detected with anti-GST antibody, and anti-HA antibody was used to detect the expression of TRIM21. ( F ) FLAG-HBV DNA Pol and HA-TRIM21 were transfected into Huh7 cells; 36h after transfection, the mouse anti-FLAG TRITC-conjugated monoclonal antibody and rabbit anti-HA FITC-conjugated polyclonal antibody were used to stain the cells, and the DAPI were used to stain the nuclei. Then, confocal microscopy images were collected and analyzed for the colocalization of HBV DNA Pol and TRIM21. The scale is 10 μm. Data are representative of three independent experiments with three replicates each.
    Figure Legend Snippet: TRIM21 interacts with HBV DNA Pol. ( A ) Huh7 cells were transfected with FLAG-HBV DNA Pol and vector control, and FLAG affinity beads were used to precipitate all proteins that might interact with HBV DNA Pol. The interacting proteins of HBV DNA Pol were screened by mass spectrometry. Some potential interacting partners of HBV DNA Pol are listed. Red marks the target protein, blue marks proteins that have been reported to interact with HBV DNA Pol. Data are representative of two independent experiments. ( B ) The FLAG-HBV DNA Pol expression plasmid was transfected into Huh7 cells. After 36h, a co-IP assay was carried out with anti-FLAG antibody and control IgG antibody. HBV DNA Pol was then detected with anti-FLAG antibody, and anti-TRIM21 antibody was used to detect the endogenous expression of TRIM21 in Huh7 cells by Western blot. ( C ) Huh7 cells were cotransfected with FLAG-HBV DNA Pol and HA-TRIM21, and co-IP was performed as described in ( B ) to detect the expression of HBV DNA Pol and TRIM21. ( D ) Huh7 cells were transfected as described in ( C ), co-IP was performed with anti-HA antibody, and HBV DNA Pol and HA-TRIM21 expression were detected by Western blot. ( E ) GST-HBV DNA Pol and HA-TRIM21 were transfected into Huh7 cells, 36h after transfection, GST beads were used to pull down GST-Pol with its interacting proteins. HBV DNA Pol was detected with anti-GST antibody, and anti-HA antibody was used to detect the expression of TRIM21. ( F ) FLAG-HBV DNA Pol and HA-TRIM21 were transfected into Huh7 cells; 36h after transfection, the mouse anti-FLAG TRITC-conjugated monoclonal antibody and rabbit anti-HA FITC-conjugated polyclonal antibody were used to stain the cells, and the DAPI were used to stain the nuclei. Then, confocal microscopy images were collected and analyzed for the colocalization of HBV DNA Pol and TRIM21. The scale is 10 μm. Data are representative of three independent experiments with three replicates each.

    Techniques Used: Transfection, Plasmid Preparation, Mass Spectrometry, Expressing, Co-Immunoprecipitation Assay, Western Blot, Staining, Confocal Microscopy

    TRIM21 restricts HBV DNA replication. ( A ) Huh7 cells were cotransfected with pHBV1.3 and pcDNA3 or TRIM21 plasmid or pSilencer-NC or shR-TRIM21. qPCR was used to detect HBV DNA. ( B ) HepG2.2.15 cells were transfected with pcDNA3, TRIM21, pSilencer-NC or shR-TRIM21. HBV DNA was detected as described in ( A ). ( C ) HepG2.2.15 cells were transfected with pcDNA3 or TRIM21, and Southern blot was used to detect the cellular replicative intermediate DNA (RI-DNA). ( D ) Huh7 cells were transfected with pHBV1.3, pcDNA3 (left panel) or pTRIM21 (right panel) and ubiquitin or K48R or K63R. HBV DNA was detected by qPCR. ( E ) Except for pHBV1.3, HepG2.2.15 cells were transfected as described in ( D ), and the relative levels of HBV DNA were detected in HepG2.2.15 cells. ( F ) Huh7 cells were cotransfected with pHBV1.3 and pcDNA3 or TRIM21-ΔSPRY, and the HBV DNA level was detected by qPCR. ( G ) Except for pHBV1.3, HepG2.2.15 cells were transfected as described in ( F ). ( H ) HepG2.2.15 cells were transfected with pcDNA3 or TRIM21-ΔSPRY, and RI-DNA was examined by Southern blot. Data are representative of three independent experiments with three replicates each. The data represent the means ± S.D. * P
    Figure Legend Snippet: TRIM21 restricts HBV DNA replication. ( A ) Huh7 cells were cotransfected with pHBV1.3 and pcDNA3 or TRIM21 plasmid or pSilencer-NC or shR-TRIM21. qPCR was used to detect HBV DNA. ( B ) HepG2.2.15 cells were transfected with pcDNA3, TRIM21, pSilencer-NC or shR-TRIM21. HBV DNA was detected as described in ( A ). ( C ) HepG2.2.15 cells were transfected with pcDNA3 or TRIM21, and Southern blot was used to detect the cellular replicative intermediate DNA (RI-DNA). ( D ) Huh7 cells were transfected with pHBV1.3, pcDNA3 (left panel) or pTRIM21 (right panel) and ubiquitin or K48R or K63R. HBV DNA was detected by qPCR. ( E ) Except for pHBV1.3, HepG2.2.15 cells were transfected as described in ( D ), and the relative levels of HBV DNA were detected in HepG2.2.15 cells. ( F ) Huh7 cells were cotransfected with pHBV1.3 and pcDNA3 or TRIM21-ΔSPRY, and the HBV DNA level was detected by qPCR. ( G ) Except for pHBV1.3, HepG2.2.15 cells were transfected as described in ( F ). ( H ) HepG2.2.15 cells were transfected with pcDNA3 or TRIM21-ΔSPRY, and RI-DNA was examined by Southern blot. Data are representative of three independent experiments with three replicates each. The data represent the means ± S.D. * P

    Techniques Used: Plasmid Preparation, Real-time Polymerase Chain Reaction, Transfection, Southern Blot

    A proposed model describing the role of TRIM21 in the regulation of HBV DNA Pol and HBV DNA replication. HBV-encoded DNA Pol forms a complex with pgRNA and is packaged into a new viral particle by core protein. Some particles reenter into the nucleus and others are enveloped and released from cells for reinfection. Thus, HBV DNA Pol is necessary for HBV replication. Here, we show that TRIM21 mainly promotes the ubiquitination and degradation of HBV DNA Pol, which reduces HBV DNA replication.
    Figure Legend Snippet: A proposed model describing the role of TRIM21 in the regulation of HBV DNA Pol and HBV DNA replication. HBV-encoded DNA Pol forms a complex with pgRNA and is packaged into a new viral particle by core protein. Some particles reenter into the nucleus and others are enveloped and released from cells for reinfection. Thus, HBV DNA Pol is necessary for HBV replication. Here, we show that TRIM21 mainly promotes the ubiquitination and degradation of HBV DNA Pol, which reduces HBV DNA replication.

    Techniques Used:

    TRIM21 promotes the degradation of HBV DNA Pol through the ubiquitin proteasome pathway via its RING domain. ( A ) Huh7 cells were transfected with FLAG-HBV DNA Pol expression plasmid. After 30 h, the cells were treated with 20 μM MG132 for 8 h, and Western blot was used to detect the protein level of HBV DNA Pol. ( B ) The cells were transfected as described in ( A ) except for using 0, 25 or 50 μM chloroquine to treat the cells for 6h. ( C ) FLAG-HBV DNA Pol was cotransfected with pcDNA3/pTRIM21 (left panel) or pSilencer-NC/shR-TRIM21 (right panel) in Huh7 cells, and MG132 treatment conditions were the same as in ( A ). Then, Western blot was used to detect the protein level of HBV DNA Pol. ( D ) Huh7 cells were cotransfected with FLAG-HBV DNA Pol and pcDNA3 (left panel)/pSilencer-NC (right panel) and treated with MG132 as described in ( A ), or cotransfected with pTRIM21 (left panel)/shR-TRIM21 (right panel). The protein level of HBV DNA Pol was analyzed by Western blot. ( E ) Huh7 cells were transfected with TRIM21 overexpression plasmid, and MG132 treatment was the same as in ( A ). Then, Western blot was used to detect the protein level of TRIM21. ( F ) The same as in ( B ), except for using chloroquine, and the cells were transfected as described in ( E ). ( G ) Huh7 cells were cotransfected with FLAG-HBV DNA Pol and pcDNA3 or HA-TRIM21, and the cells were treated with 10 μM MG132. Subsequently, anti-FLAG antibody was used to precipitate the proteins for ubiquitination analysis. ( H ) The same as in ( G ), except for pSilencer-NC and shR-TRIM21, which were separately cotransfected with FLAG-HBV DNA Pol. ( I ) Huh7 cells were cotransfected FLAG-HBV DNA Pol with wild-type TRIM21, TRIM21-ΔRING or pcDNA3. The ubiquitin antibody was used to detect the ubiquitination level of HBV DNA Pol, and the anti-FLAG antibody was used to detect the protein level. Data are representative of three independent experiments with three replicates each.
    Figure Legend Snippet: TRIM21 promotes the degradation of HBV DNA Pol through the ubiquitin proteasome pathway via its RING domain. ( A ) Huh7 cells were transfected with FLAG-HBV DNA Pol expression plasmid. After 30 h, the cells were treated with 20 μM MG132 for 8 h, and Western blot was used to detect the protein level of HBV DNA Pol. ( B ) The cells were transfected as described in ( A ) except for using 0, 25 or 50 μM chloroquine to treat the cells for 6h. ( C ) FLAG-HBV DNA Pol was cotransfected with pcDNA3/pTRIM21 (left panel) or pSilencer-NC/shR-TRIM21 (right panel) in Huh7 cells, and MG132 treatment conditions were the same as in ( A ). Then, Western blot was used to detect the protein level of HBV DNA Pol. ( D ) Huh7 cells were cotransfected with FLAG-HBV DNA Pol and pcDNA3 (left panel)/pSilencer-NC (right panel) and treated with MG132 as described in ( A ), or cotransfected with pTRIM21 (left panel)/shR-TRIM21 (right panel). The protein level of HBV DNA Pol was analyzed by Western blot. ( E ) Huh7 cells were transfected with TRIM21 overexpression plasmid, and MG132 treatment was the same as in ( A ). Then, Western blot was used to detect the protein level of TRIM21. ( F ) The same as in ( B ), except for using chloroquine, and the cells were transfected as described in ( E ). ( G ) Huh7 cells were cotransfected with FLAG-HBV DNA Pol and pcDNA3 or HA-TRIM21, and the cells were treated with 10 μM MG132. Subsequently, anti-FLAG antibody was used to precipitate the proteins for ubiquitination analysis. ( H ) The same as in ( G ), except for pSilencer-NC and shR-TRIM21, which were separately cotransfected with FLAG-HBV DNA Pol. ( I ) Huh7 cells were cotransfected FLAG-HBV DNA Pol with wild-type TRIM21, TRIM21-ΔRING or pcDNA3. The ubiquitin antibody was used to detect the ubiquitination level of HBV DNA Pol, and the anti-FLAG antibody was used to detect the protein level. Data are representative of three independent experiments with three replicates each.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Western Blot, Over Expression

    TRIM21 interacts with the TP domain of HBV DNA Pol via its SPRY domain. ( A ) Schematic map of the deletion mutants of HBV DNA Pol. ( B ) HA-TRIM21 was cotransfected with FLAG-HBV DNA Pol, ΔTP, ΔRT or ΔRH. Anti-HA antibody was used for immunoprecipitation, and anti-FLAG antibody was used to detect the expression of HBV DNA Pol. ( C ) TRIM21 and its truncation mutants. ( D ) Huh7 cells were cotransfected with Myc-HBV DNA Pol and FLAG-TRIM21 or its truncation mutants, and co-IP was used to analyze the interaction of Myc-HBV DNA Pol with FLAG-TRIM21 or its truncation mutants. ( E ) GST-HBV DNA Pol and HA-ΔSPRY were transfected into Huh7 cells, and 36 h post transfection, GST beads were used to pull down GST-Pol with its interacting proteins. HBV DNA Pol was detected with anti-GST antibody, and anti-HA antibody was used to detect the expression of ΔSPRY. Data are representative of three independent experiments with three replicates each.
    Figure Legend Snippet: TRIM21 interacts with the TP domain of HBV DNA Pol via its SPRY domain. ( A ) Schematic map of the deletion mutants of HBV DNA Pol. ( B ) HA-TRIM21 was cotransfected with FLAG-HBV DNA Pol, ΔTP, ΔRT or ΔRH. Anti-HA antibody was used for immunoprecipitation, and anti-FLAG antibody was used to detect the expression of HBV DNA Pol. ( C ) TRIM21 and its truncation mutants. ( D ) Huh7 cells were cotransfected with Myc-HBV DNA Pol and FLAG-TRIM21 or its truncation mutants, and co-IP was used to analyze the interaction of Myc-HBV DNA Pol with FLAG-TRIM21 or its truncation mutants. ( E ) GST-HBV DNA Pol and HA-ΔSPRY were transfected into Huh7 cells, and 36 h post transfection, GST beads were used to pull down GST-Pol with its interacting proteins. HBV DNA Pol was detected with anti-GST antibody, and anti-HA antibody was used to detect the expression of ΔSPRY. Data are representative of three independent experiments with three replicates each.

    Techniques Used: Immunoprecipitation, Expressing, Co-Immunoprecipitation Assay, Transfection

    37) Product Images from "BUBR1 recruits PP2A via the B56 family of targeting subunits to promote chromosome congression"

    Article Title: BUBR1 recruits PP2A via the B56 family of targeting subunits to promote chromosome congression

    Journal: Biology Open

    doi: 10.1242/bio.20134051

    Isolation and characterization of BUBR1 as a novel direct binding partner of all B56 subunits. ( A , D , E , G ) Yeast two-hybrid assay. The interaction between BUBR1 and B56 subunits was evaluated by colony growth as well as X-gal assay. For C (top panel), structural motifs of human BUBR1 with a summary of interactions between a series of deletion mutants of BUBR1 and B56δ are shown. ( B ) HeLa cell lysates transiently expressing HA-B56δ and Myc-BUBR1 were subjected to immunoprecipitation analysis with antibodies against HA-epitope. ( F ) Alignment of the putative second B56-binding sequence of BUBR1 between different species. The changes in amino acid residues to generate the indicated point mutants of BUBR1 for yeast two-hybrid assay (G) are shown.
    Figure Legend Snippet: Isolation and characterization of BUBR1 as a novel direct binding partner of all B56 subunits. ( A , D , E , G ) Yeast two-hybrid assay. The interaction between BUBR1 and B56 subunits was evaluated by colony growth as well as X-gal assay. For C (top panel), structural motifs of human BUBR1 with a summary of interactions between a series of deletion mutants of BUBR1 and B56δ are shown. ( B ) HeLa cell lysates transiently expressing HA-B56δ and Myc-BUBR1 were subjected to immunoprecipitation analysis with antibodies against HA-epitope. ( F ) Alignment of the putative second B56-binding sequence of BUBR1 between different species. The changes in amino acid residues to generate the indicated point mutants of BUBR1 for yeast two-hybrid assay (G) are shown.

    Techniques Used: Isolation, Binding Assay, Y2H Assay, Expressing, Immunoprecipitation, Sequencing

    The B56:BUBR1 interaction is required for chromosome congression. ( A ) Schematic experimental procedure. Asynchronously growing HeLa cells were transfected twice with a 24-hour interval using control non-silencing or two different siRNAs against BUBR1 (100 nM). During second transfection, the indicated expression vectors encoding siRNA-insensitive LAP-tagged BUBR1 were co-transfected. Forty-eight hours after initial transfection, cells were treated with MG132 for 3 hours, and subjected to immunofluorescence analysis using antibodies against α-tubulin and DAPI to visualize the mitotic spindles and chromosomes, respectively. ( B , C ) Quantification results of chromosome congression defects in HeLa cells ( n > 100 cells counted for each condition) expressing the indicated LAP-BUBR1. Immunoblot of expressed LAP-BUBR1 were shown (top panels). Scale bar = 5 µm. ( D ) Lysates from HeLa cells transiently expressing HA-B56α and LAP-BUBR1 as indicated were subjected to immunoprecipitation analysis with antibodies against GFP.
    Figure Legend Snippet: The B56:BUBR1 interaction is required for chromosome congression. ( A ) Schematic experimental procedure. Asynchronously growing HeLa cells were transfected twice with a 24-hour interval using control non-silencing or two different siRNAs against BUBR1 (100 nM). During second transfection, the indicated expression vectors encoding siRNA-insensitive LAP-tagged BUBR1 were co-transfected. Forty-eight hours after initial transfection, cells were treated with MG132 for 3 hours, and subjected to immunofluorescence analysis using antibodies against α-tubulin and DAPI to visualize the mitotic spindles and chromosomes, respectively. ( B , C ) Quantification results of chromosome congression defects in HeLa cells ( n > 100 cells counted for each condition) expressing the indicated LAP-BUBR1. Immunoblot of expressed LAP-BUBR1 were shown (top panels). Scale bar = 5 µm. ( D ) Lysates from HeLa cells transiently expressing HA-B56α and LAP-BUBR1 as indicated were subjected to immunoprecipitation analysis with antibodies against GFP.

    Techniques Used: Transfection, Expressing, Immunofluorescence, Immunoprecipitation

    38) Product Images from "Comprehensive analysis of heterotrimeric G-protein complex diversity and their interactions with GPCRs in solution"

    Article Title: Comprehensive analysis of heterotrimeric G-protein complex diversity and their interactions with GPCRs in solution

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1417573112

    Functional interaction of the NTR1 WT receptor and its evolved mutants with G protein. ( A ) [ 35 S]GTPγS assay of the NTR1 WT receptor, the stabilized mutant TM86V and TM86V with restored E/DRY motif (TM86V L167R). The amount of [ 35 S]GTPγS bound to the G protein (α i1 β 1 γ 1 ) in the presence or absence of the agonist NT (20 µM) is shown. The signals correspond to the average of two or three (NTRI) experiments performed in parallel from independent GPCR expressions. Error bars represent SDs. ( B ) Results of coimmunoprecipitation experiments of G protein (α i1 β 1 HA-γ 1 ) and NTR1 WT or TM86V L167R. After incubation of the solubilized G protein with anti-HA beads, the beads were incubated with solubilized GPCR. The presence of solubilized proteins before incubation with the beads (load) and the GPCR/G protein bound to the beads as a function of the presence of G protein, ligand (20 µM NT) and GTPγS (750 µM) is shown by Western blot (bound). ( C ) Coimmunoprecipitation experiments comparing TM86V with TM86V L167R, carried out as described for B .
    Figure Legend Snippet: Functional interaction of the NTR1 WT receptor and its evolved mutants with G protein. ( A ) [ 35 S]GTPγS assay of the NTR1 WT receptor, the stabilized mutant TM86V and TM86V with restored E/DRY motif (TM86V L167R). The amount of [ 35 S]GTPγS bound to the G protein (α i1 β 1 γ 1 ) in the presence or absence of the agonist NT (20 µM) is shown. The signals correspond to the average of two or three (NTRI) experiments performed in parallel from independent GPCR expressions. Error bars represent SDs. ( B ) Results of coimmunoprecipitation experiments of G protein (α i1 β 1 HA-γ 1 ) and NTR1 WT or TM86V L167R. After incubation of the solubilized G protein with anti-HA beads, the beads were incubated with solubilized GPCR. The presence of solubilized proteins before incubation with the beads (load) and the GPCR/G protein bound to the beads as a function of the presence of G protein, ligand (20 µM NT) and GTPγS (750 µM) is shown by Western blot (bound). ( C ) Coimmunoprecipitation experiments comparing TM86V with TM86V L167R, carried out as described for B .

    Techniques Used: Functional Assay, GTPγS Binding Assay, Mutagenesis, Incubation, Western Blot

    Identification of the interaction preferences between Gα i1 βγ heterotrimer combinations and NTR1 or TM86V L167R. ( A ) Heterotrimer combinations consisting of α i1 , β 1–4 and HA-γ 1–13 were tested for their interaction with NTR1 WT receptor in the presence of 20 µM NT in a coimmunoprecipitation experiment directed against the HA tag of γ. The results are presented in form of Western blots against NTR1 and the α i1 -subunit. As a visual guide to assess the potency of GPCR/G-protein complex formation, the ratio of signal intensities between GPCR and α i1 -subunit are given below the blots. Bar heights indicate the interaction preference of a given G-protein combination for the GPCR in question, with higher being better. ( B ) Interaction studies for TM86V L167R as in A .
    Figure Legend Snippet: Identification of the interaction preferences between Gα i1 βγ heterotrimer combinations and NTR1 or TM86V L167R. ( A ) Heterotrimer combinations consisting of α i1 , β 1–4 and HA-γ 1–13 were tested for their interaction with NTR1 WT receptor in the presence of 20 µM NT in a coimmunoprecipitation experiment directed against the HA tag of γ. The results are presented in form of Western blots against NTR1 and the α i1 -subunit. As a visual guide to assess the potency of GPCR/G-protein complex formation, the ratio of signal intensities between GPCR and α i1 -subunit are given below the blots. Bar heights indicate the interaction preference of a given G-protein combination for the GPCR in question, with higher being better. ( B ) Interaction studies for TM86V L167R as in A .

    Techniques Used: Western Blot

    39) Product Images from "IKK phosphorylates Huntingtin and targets it for degradation by the proteasome and lysosome"

    Article Title: IKK phosphorylates Huntingtin and targets it for degradation by the proteasome and lysosome

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200909067

    Phosphorylation of unexpanded polyQ Httex1p and 586aa Htt is associated with its reduced abundance in cell culture. (A) Levels of unexpanded polyQ Httex1p are reduced with overexpression of IKK-β; this effect is inhibited with expansion of the polyQ repeat. 25QP-H4 or 46QP-H4 was cotransfected with myc-actin and with vector or IKK-β into St14A cells. Cells were treated for 16 h with DMSO, 100 nM epoxomicin in DMSO, or 20 mM ammonium chloride/100 µM leupeptin in DMSO. Lysates were subjected to filter-retardation assay and Western analysis using anti-myc to detect myc-actin, and anti-HA to detect Httex1p. (B) IKK-β overexpression reduces levels of unexpanded polyQ Httex1p in the presence of proteasome or lysosome inhibition. Scion software was used to quantitate triplicate levels of 25QP-H4 from the experiment represented in A, normalized to levels of myc-actin transfection control, within each treatment group: control, epoxomicin, or ammonium chloride/leupeptin. (C) Mimicking phosphorylation of unexpanded polyQ Httex1p reduces its abundance in cell culture; this effect is reduced with expansion of the polyQ repeat. 25QP-H4 or 46QP-H4, wt control or QEE, EE, AA, or 3R were cotransfected with myc-actin into St14A cells. Cells and lysates were treated as in A. (D) Levels of phosphorylated unexpanded polyQ 586aa Htt accumulate with inhibition of the proteasome or the lysosome; phosphorylation is reduced with expansion of the polyQ repeat. 15Q or 128Q 586aa Htt constructs were cotransfected into St14A cells with myc-actin and with vector or IKK-β. Cells were treated for 4 h with DMSO or 100 nM epoxomicin in DMSO (to eliminate any possible effect on the lysosome by epoxomicin), or for 16 h with water or 20 mM ammonium chloride/100 µM leupeptin in water. Lysates were subjected to filter-retardation assay and Western analysis using anti-myc, anti–S13-P, and anti-Htt 3B5H10.
    Figure Legend Snippet: Phosphorylation of unexpanded polyQ Httex1p and 586aa Htt is associated with its reduced abundance in cell culture. (A) Levels of unexpanded polyQ Httex1p are reduced with overexpression of IKK-β; this effect is inhibited with expansion of the polyQ repeat. 25QP-H4 or 46QP-H4 was cotransfected with myc-actin and with vector or IKK-β into St14A cells. Cells were treated for 16 h with DMSO, 100 nM epoxomicin in DMSO, or 20 mM ammonium chloride/100 µM leupeptin in DMSO. Lysates were subjected to filter-retardation assay and Western analysis using anti-myc to detect myc-actin, and anti-HA to detect Httex1p. (B) IKK-β overexpression reduces levels of unexpanded polyQ Httex1p in the presence of proteasome or lysosome inhibition. Scion software was used to quantitate triplicate levels of 25QP-H4 from the experiment represented in A, normalized to levels of myc-actin transfection control, within each treatment group: control, epoxomicin, or ammonium chloride/leupeptin. (C) Mimicking phosphorylation of unexpanded polyQ Httex1p reduces its abundance in cell culture; this effect is reduced with expansion of the polyQ repeat. 25QP-H4 or 46QP-H4, wt control or QEE, EE, AA, or 3R were cotransfected with myc-actin into St14A cells. Cells and lysates were treated as in A. (D) Levels of phosphorylated unexpanded polyQ 586aa Htt accumulate with inhibition of the proteasome or the lysosome; phosphorylation is reduced with expansion of the polyQ repeat. 15Q or 128Q 586aa Htt constructs were cotransfected into St14A cells with myc-actin and with vector or IKK-β. Cells were treated for 4 h with DMSO or 100 nM epoxomicin in DMSO (to eliminate any possible effect on the lysosome by epoxomicin), or for 16 h with water or 20 mM ammonium chloride/100 µM leupeptin in water. Lysates were subjected to filter-retardation assay and Western analysis using anti-myc, anti–S13-P, and anti-Htt 3B5H10.

    Techniques Used: Cell Culture, Over Expression, Plasmid Preparation, Western Blot, Inhibition, Software, Transfection, Construct

    40) Product Images from "The Ciliopathy Protein CC2D2A Associates with NINL and Functions in RAB8-MICAL3-Regulated Vesicle Trafficking"

    Article Title: The Ciliopathy Protein CC2D2A Associates with NINL and Functions in RAB8-MICAL3-Regulated Vesicle Trafficking

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1005575

    CC2D2A associates with NINL. ( a ) Yeast two-hybrid interaction assays were performed with different fragments of CC2D2A fused to the GAL4 DNA binding domain (BD) and full length NINL isoform A and B, fused to the GAL4 activation domain (AD). Activation of the reporter genes, which indicates a physical interaction, was dependent on coiled-coil (CC) domains 1 and 2 of CC2D2A and either NINL isoform A or B. ( b ) The top panel of the immunoblot (IB) shows that FLAG-tagged CC2D2A, but not the FLAG-tagged LRRK2 that was included as a negative control, was co-precipitated with HA-tagged NINL isoform B using a rat monoclonal antibody directed against the HA-epitope. Protein input is shown in the lower panel; anti-HA precipitates are shown in the middle panel. ( c ) In a reciprocal experiment, HA-tagged NINL isoB was co-precipitated with FLAG-tagged CC2D2A, but not with FLAG-tagged LRRK2. Protein input is shown in the lower panel; anti-FLAG precipitates are shown in the middle panel.
    Figure Legend Snippet: CC2D2A associates with NINL. ( a ) Yeast two-hybrid interaction assays were performed with different fragments of CC2D2A fused to the GAL4 DNA binding domain (BD) and full length NINL isoform A and B, fused to the GAL4 activation domain (AD). Activation of the reporter genes, which indicates a physical interaction, was dependent on coiled-coil (CC) domains 1 and 2 of CC2D2A and either NINL isoform A or B. ( b ) The top panel of the immunoblot (IB) shows that FLAG-tagged CC2D2A, but not the FLAG-tagged LRRK2 that was included as a negative control, was co-precipitated with HA-tagged NINL isoform B using a rat monoclonal antibody directed against the HA-epitope. Protein input is shown in the lower panel; anti-HA precipitates are shown in the middle panel. ( c ) In a reciprocal experiment, HA-tagged NINL isoB was co-precipitated with FLAG-tagged CC2D2A, but not with FLAG-tagged LRRK2. Protein input is shown in the lower panel; anti-FLAG precipitates are shown in the middle panel.

    Techniques Used: Binding Assay, Activation Assay, Negative Control

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    Radio Immunoprecipitation:

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    XTT Assay:

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    Protease Inhibitor:

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    Incubation:

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    Cell Culture:

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    Viability Assay:

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