confluent cos 1 cells  (Thermo Fisher)


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    Structured Review

    Thermo Fisher confluent cos 1 cells
    KLF5 colocalizes with SMURF2. <t>COS-1</t> cells were transfected with HA-KLF5 and Myc-SMURF2, treated with MG132 to stabilize HA-KLF5, and stained with chicken anti-HA and rabbit anti-Myc, followed by FITC-conjugated donkey α-chicken and Cy5-conjugated
    Confluent Cos 1 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 5195 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "The E3 Ubiquitin Ligase SMAD Ubiquitination Regulatory Factor 2 Negatively Regulates Kr?ppel-like Factor 5 Protein *"

    Article Title: The E3 Ubiquitin Ligase SMAD Ubiquitination Regulatory Factor 2 Negatively Regulates Kr?ppel-like Factor 5 Protein *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.258707

    KLF5 colocalizes with SMURF2. COS-1 cells were transfected with HA-KLF5 and Myc-SMURF2, treated with MG132 to stabilize HA-KLF5, and stained with chicken anti-HA and rabbit anti-Myc, followed by FITC-conjugated donkey α-chicken and Cy5-conjugated
    Figure Legend Snippet: KLF5 colocalizes with SMURF2. COS-1 cells were transfected with HA-KLF5 and Myc-SMURF2, treated with MG132 to stabilize HA-KLF5, and stained with chicken anti-HA and rabbit anti-Myc, followed by FITC-conjugated donkey α-chicken and Cy5-conjugated

    Techniques Used: Transfection, Staining

    SMURF2 inhibits the transcriptional and pro-proliferative activities of KLF5. A , depletion of SMURF2 increases the expression of KLF5 target genes. COS-1 cells were transfected with either control siRNA or the Trilencer SMURF2 siRNA mixture from Origene
    Figure Legend Snippet: SMURF2 inhibits the transcriptional and pro-proliferative activities of KLF5. A , depletion of SMURF2 increases the expression of KLF5 target genes. COS-1 cells were transfected with either control siRNA or the Trilencer SMURF2 siRNA mixture from Origene

    Techniques Used: Expressing, Transfection

    2) Product Images from "A large-scale functional screen identifies Nova1 and Ncoa3 as regulators of neuronal miRNA function"

    Article Title: A large-scale functional screen identifies Nova1 and Ncoa3 as regulators of neuronal miRNA function

    Journal: The EMBO Journal

    doi: 10.15252/embj.201490643

    Identification of 12 RNA-binding proteins required for miR-134 repressive function in primary neurons using siRNA-based screening
    Figure Legend Snippet: Identification of 12 RNA-binding proteins required for miR-134 repressive function in primary neurons using siRNA-based screening

    Techniques Used: RNA Binding Assay

    3) Product Images from "A large-scale functional screen identifies Nova1 and Ncoa3 as regulators of neuronal miRNA function"

    Article Title: A large-scale functional screen identifies Nova1 and Ncoa3 as regulators of neuronal miRNA function

    Journal: The EMBO Journal

    doi: 10.15252/embj.201490643

    Identification of 12 RNA-binding proteins required for miR-134 repressive function in primary neurons using siRNA-based screening
    Figure Legend Snippet: Identification of 12 RNA-binding proteins required for miR-134 repressive function in primary neurons using siRNA-based screening

    Techniques Used: RNA Binding Assay

    4) Product Images from "Human Negative Elongation Factor Activates Transcription and Regulates Alternative Transcription Initiation *"

    Article Title: Human Negative Elongation Factor Activates Transcription and Regulates Alternative Transcription Initiation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.084285

    Exon array-based profiling of gene expression in NELF knockdown cells. A , Western blot analysis of NELF expression in T47D cells transfected with siRNA oligonucleotides for control luciferase (siControl) or the individual NELF subunits. α-Tubulin
    Figure Legend Snippet: Exon array-based profiling of gene expression in NELF knockdown cells. A , Western blot analysis of NELF expression in T47D cells transfected with siRNA oligonucleotides for control luciferase (siControl) or the individual NELF subunits. α-Tubulin

    Techniques Used: Expressing, Western Blot, Transfection, Luciferase

    NELF depletion increases total histone density and decreases activation-associated histone modifications at NELF target genes. T47D cells transfected with control and NELF-E siRNA oligonucleotides were used for ChIP with antibodies recognizing total histone
    Figure Legend Snippet: NELF depletion increases total histone density and decreases activation-associated histone modifications at NELF target genes. T47D cells transfected with control and NELF-E siRNA oligonucleotides were used for ChIP with antibodies recognizing total histone

    Techniques Used: Activation Assay, Transfection, Chromatin Immunoprecipitation

    NELF is directly involved in transcription of a number of cell cycle-associated genes. A , real time PCR analysis of gene expression in control and NELF knockdown cells. T47D cells were transfected with siRNA oligonucleotides for 4 days. Total RNA was
    Figure Legend Snippet: NELF is directly involved in transcription of a number of cell cycle-associated genes. A , real time PCR analysis of gene expression in control and NELF knockdown cells. T47D cells were transfected with siRNA oligonucleotides for 4 days. Total RNA was

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

    NELF depletion delays cell cycle progression. A , effect of NELF knockdown on cell proliferation. T47D cells were transfected with siRNA oligonucleotides against the individual NELF subunits, and the cell number was counted on days 2, 3, and 4 after siRNA
    Figure Legend Snippet: NELF depletion delays cell cycle progression. A , effect of NELF knockdown on cell proliferation. T47D cells were transfected with siRNA oligonucleotides against the individual NELF subunits, and the cell number was counted on days 2, 3, and 4 after siRNA

    Techniques Used: Transfection

    In vivo association of NELF-C with NELF-regulated genes. A , recruitment of NELF-C to the promoter-proximal regions of NELF targets. Sonicated chromatin extract from T47D cells was immunoprecipitated with IgG or NELF-C antiserum. Association of NELF-C
    Figure Legend Snippet: In vivo association of NELF-C with NELF-regulated genes. A , recruitment of NELF-C to the promoter-proximal regions of NELF targets. Sonicated chromatin extract from T47D cells was immunoprecipitated with IgG or NELF-C antiserum. Association of NELF-C

    Techniques Used: In Vivo, Sonication, Immunoprecipitation

    5) Product Images from "Signaling through ShcA Is Required for Transforming Growth Factor β- and Neu/ErbB-2-Induced Breast Cancer Cell Motility and Invasion "

    Article Title: Signaling through ShcA Is Required for Transforming Growth Factor β- and Neu/ErbB-2-Induced Breast Cancer Cell Motility and Invasion

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01734-07

    Transient siRNA-mediated knockdown of the ShcA adaptor protein in Neu/ErbB-2-expressing breast cancer explants. (A) Immunoblot analysis of Neu-NT-expressing NMuMG breast cancer cells treated with a scrambled control siRNA (C) or a mixture of three siRNAs
    Figure Legend Snippet: Transient siRNA-mediated knockdown of the ShcA adaptor protein in Neu/ErbB-2-expressing breast cancer explants. (A) Immunoblot analysis of Neu-NT-expressing NMuMG breast cancer cells treated with a scrambled control siRNA (C) or a mixture of three siRNAs

    Techniques Used: Expressing

    6) Product Images from "PED is overexpressed and mediates TRAIL resistance in human non-small cell lung cancer"

    Article Title: PED is overexpressed and mediates TRAIL resistance in human non-small cell lung cancer

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2008.00283.x

    Effects of PED on caspase activation. (A) CALU-1 cells were transfected with PED or control siRNA for 72 hrs and then treated with superkiller TRAIL for the indicated times. Lysates were examined by Western blotting with anti-caspase 8 or anti-PARP antibodies. Cleavage of caspase 8 and PARP was detected at a greater amount in CALU-1 cells transfected with PED siRNA. β-Actin was used as the loading control. (B) PED cDNA (PED-Myc) was transiently transfected in H460 cells and cells were analysed for caspase 8 and PARP activation as previously described or for or for cell viability (D) as indicated. (C) Western blot analysis of PED expression revealed that transfection increased PED expression levels in H460 cells. β-Actin was used as the loading control. Representative blots are shown.
    Figure Legend Snippet: Effects of PED on caspase activation. (A) CALU-1 cells were transfected with PED or control siRNA for 72 hrs and then treated with superkiller TRAIL for the indicated times. Lysates were examined by Western blotting with anti-caspase 8 or anti-PARP antibodies. Cleavage of caspase 8 and PARP was detected at a greater amount in CALU-1 cells transfected with PED siRNA. β-Actin was used as the loading control. (B) PED cDNA (PED-Myc) was transiently transfected in H460 cells and cells were analysed for caspase 8 and PARP activation as previously described or for or for cell viability (D) as indicated. (C) Western blot analysis of PED expression revealed that transfection increased PED expression levels in H460 cells. β-Actin was used as the loading control. Representative blots are shown.

    Techniques Used: Activation Assay, Transfection, Western Blot, Expressing

    Down-regulation of PED restores TRAIL sensitivity in CALU-1 cells. (A) PED siRNA or a control oligo were transiently transfected in CALU-1 cells in the presence or absence of PED-Myc cDNA. Cells were incubated for 48 or 72 hrs and analysed by Western blotting. The PED siRNA duplex suppressed both exogenous and endogenous PED expression, whereas control siRNA had no effects. (B) PED siRNA effects in A459 and A549 cells. PEDsi RNA, transfected in NSCLC cells was able to reduce PED expression levels (right panel) and induce an increase in TRAIL sensitivity (left panel), as assessed by flow cytometry. Mean ± SD of two independent experiments in duplicate. (C) c-FLIPL siRNA or PED siRNA were transfected as described in Methods. Cells were analysed for c-FLIP expression after 72 hrs incubation. c-FLIPL siRNA but not PED siRNA was able to reduce c-FLIPL expression Effects of silencing PED and c-FLIPL on TRAIL-induced cell death: CALU-1 cells were transfected with siRNA for PED, c-FLIPL or control for 48 hrs, after which cells were trypsinized, plated in 96-well plates in triplicate and further incubated with superkiller TRAIL for 24 hrs. Metabolically active cells were then detected as indicated in the Methods. Mean ± SD of four independent experiments in duplicate. Down-regulation of PED, but not cFLIPL, was responsible for increased sensitivity of CALU-1 cells to TRAIL-mediated cell death.
    Figure Legend Snippet: Down-regulation of PED restores TRAIL sensitivity in CALU-1 cells. (A) PED siRNA or a control oligo were transiently transfected in CALU-1 cells in the presence or absence of PED-Myc cDNA. Cells were incubated for 48 or 72 hrs and analysed by Western blotting. The PED siRNA duplex suppressed both exogenous and endogenous PED expression, whereas control siRNA had no effects. (B) PED siRNA effects in A459 and A549 cells. PEDsi RNA, transfected in NSCLC cells was able to reduce PED expression levels (right panel) and induce an increase in TRAIL sensitivity (left panel), as assessed by flow cytometry. Mean ± SD of two independent experiments in duplicate. (C) c-FLIPL siRNA or PED siRNA were transfected as described in Methods. Cells were analysed for c-FLIP expression after 72 hrs incubation. c-FLIPL siRNA but not PED siRNA was able to reduce c-FLIPL expression Effects of silencing PED and c-FLIPL on TRAIL-induced cell death: CALU-1 cells were transfected with siRNA for PED, c-FLIPL or control for 48 hrs, after which cells were trypsinized, plated in 96-well plates in triplicate and further incubated with superkiller TRAIL for 24 hrs. Metabolically active cells were then detected as indicated in the Methods. Mean ± SD of four independent experiments in duplicate. Down-regulation of PED, but not cFLIPL, was responsible for increased sensitivity of CALU-1 cells to TRAIL-mediated cell death.

    Techniques Used: Transfection, Incubation, Western Blot, Expressing, Flow Cytometry, Cytometry, Metabolic Labelling

    7) Product Images from "Plakophilin 3 mediates Rap1-dependent desmosome assembly and adherens junction maturation"

    Article Title: Plakophilin 3 mediates Rap1-dependent desmosome assembly and adherens junction maturation

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E14-05-0968

    Pkp3-dependent Rap1 activation is required for normal DP border localization. (A) Immunofluorescence staining showing the effects of EPAC activator (8-CPT-2Me-cAMP) treatment on DP border localization in control and Pkp3 KD SCC9 cells. Scale bar, 20 μm. (B) Average DP border intensity for experiments in A. Bars represent mean ± SEM. ns, p > 0.05; *** p
    Figure Legend Snippet: Pkp3-dependent Rap1 activation is required for normal DP border localization. (A) Immunofluorescence staining showing the effects of EPAC activator (8-CPT-2Me-cAMP) treatment on DP border localization in control and Pkp3 KD SCC9 cells. Scale bar, 20 μm. (B) Average DP border intensity for experiments in A. Bars represent mean ± SEM. ns, p > 0.05; *** p

    Techniques Used: Activation Assay, Immunofluorescence, Staining, Cycling Probe Technology

    Pkp3 deficiency leads to aberrant coalescence of DP at cell–cell borders. (A) Representative still images taken from the Supplemental Videos S1 and S2 showing appearance of DP at the borders at times indicated. In control cells, indicated times coincide with the wound closure. In Pkp3KD cells, white arrow indicates the closing wound starting at 14 min. White solid rectangles to the left delineate regions enlarged at the right. DP cytoplasmic and membrane particles in control and Pkp3 KD cells, respectively, were colorized as follows: pink, purple, orange, red, and blue to highlight cytoplasmic DP precursors migrating into the cell–cell border in control cells; red, dark green, orange, yellow, pink, blue, and purple to highlight membrane DP coalescing at the sites of cell–cell contacts in Pkp3KD cells; and green to highlight DP appearing, then coalescing, at the site of newly forming cell–cell border in Pkp3KD cells. Scale bar, 20 μm. (B) Graph representing average fluorescent pixel intensities of DP present at the sites of representative cell–cell contacts, measured every 5 min in A431 cells establishing new cell–cell junctions upon wound closure. Blue, green, and orange brackets indicate phases 1–3 in control cells, respectively. (C) Vertical scatter plot representing the number of DP particles present at individual borders at times indicated during live-cell imaging experiments. Number of borders analyzed: control, 16; Pkp3 KD, 18. Fewer borders were analyzed for 3-h time point, as not all border formation was followed by live-cell imaging for 3 h: control, N = 12; Pkp3 for KD, N = 17. Bars represent mean ± SEM. ns, p > 0.5, *** p
    Figure Legend Snippet: Pkp3 deficiency leads to aberrant coalescence of DP at cell–cell borders. (A) Representative still images taken from the Supplemental Videos S1 and S2 showing appearance of DP at the borders at times indicated. In control cells, indicated times coincide with the wound closure. In Pkp3KD cells, white arrow indicates the closing wound starting at 14 min. White solid rectangles to the left delineate regions enlarged at the right. DP cytoplasmic and membrane particles in control and Pkp3 KD cells, respectively, were colorized as follows: pink, purple, orange, red, and blue to highlight cytoplasmic DP precursors migrating into the cell–cell border in control cells; red, dark green, orange, yellow, pink, blue, and purple to highlight membrane DP coalescing at the sites of cell–cell contacts in Pkp3KD cells; and green to highlight DP appearing, then coalescing, at the site of newly forming cell–cell border in Pkp3KD cells. Scale bar, 20 μm. (B) Graph representing average fluorescent pixel intensities of DP present at the sites of representative cell–cell contacts, measured every 5 min in A431 cells establishing new cell–cell junctions upon wound closure. Blue, green, and orange brackets indicate phases 1–3 in control cells, respectively. (C) Vertical scatter plot representing the number of DP particles present at individual borders at times indicated during live-cell imaging experiments. Number of borders analyzed: control, 16; Pkp3 KD, 18. Fewer borders were analyzed for 3-h time point, as not all border formation was followed by live-cell imaging for 3 h: control, N = 12; Pkp3 for KD, N = 17. Bars represent mean ± SEM. ns, p > 0.5, *** p

    Techniques Used: Live Cell Imaging

    Pkp3 ablation disrupts desmosomes. (A) Western blot showing no change in the levels of indicated desmosomal and adherens junction molecules in SCC9 cells in Pkp2 and 3 siRNA knockdown. (B) Immunofluorescence showing punctate Pkp2, Dsc2, Dsg2, and Pg staining at the sites of cell–cell contacts in Pkp3 KD SCC9 cells compared with control. Scale bar, 20 μm. (C) Electron micrographs demonstrating changes in size (yellow arrows, large, single desmosomes) and morphology (red arrows, large, tandem desmosomes) of desmosomes in Pkp3 KD as compared with the control SCC9 cells. Scale bar, 1 μm, 100 nm (enlargements). (D) Scatter plots showing an increase in length (top; triangles) and width (bottom; diamonds) of individual desmosomes observed by electron microscopy in control (blue) and Pkp3 KD (red). Horizontal lines represent mean ± SEM. *** p
    Figure Legend Snippet: Pkp3 ablation disrupts desmosomes. (A) Western blot showing no change in the levels of indicated desmosomal and adherens junction molecules in SCC9 cells in Pkp2 and 3 siRNA knockdown. (B) Immunofluorescence showing punctate Pkp2, Dsc2, Dsg2, and Pg staining at the sites of cell–cell contacts in Pkp3 KD SCC9 cells compared with control. Scale bar, 20 μm. (C) Electron micrographs demonstrating changes in size (yellow arrows, large, single desmosomes) and morphology (red arrows, large, tandem desmosomes) of desmosomes in Pkp3 KD as compared with the control SCC9 cells. Scale bar, 1 μm, 100 nm (enlargements). (D) Scatter plots showing an increase in length (top; triangles) and width (bottom; diamonds) of individual desmosomes observed by electron microscopy in control (blue) and Pkp3 KD (red). Horizontal lines represent mean ± SEM. *** p

    Techniques Used: Western Blot, Immunofluorescence, Staining, Electron Microscopy

    Pkp3 mediates recruitment of soluble cytoplasmic DP to the sites of cell–cell contacts. (A) Western blot showing the difference in the presence of DP in cytoplasmic (saponin soluble) and membrane/cytoskeleton-bound (urea soluble) fractions in Pkp3 KD cells compared with control and Pkp2 KD cells. GAPDH and keratin 18 (K18) were used as loading controls for their respective fractions. (B) Western blot showing the levels of DP in the cytoplasmic (saponin soluble) cell fraction under conditions of calcium switch. GAPDH- normalized DP band intensity for each condition was determined using ImageJ. Ratio between soluble cytoplasmic DP in control and Pkp3 KD cells shows a rapid decrease in cytoplasmic DP in control cells. (C) Representative immunofluorescence images of DP appearing at cell borders after calcium switch from low to calcium concentration permissive for cell–cell junction formation, after the time indicated. “High Ca2+” represents cells that were switched overnight and are identical to steady-state conditions. White solid rectangles in the images to the left delineate areas enlarged at the right. Scale bar, 20 μm. (D) Ratio between the average fluorescence pixel intensities of cell–cell border and cytoplasmic DP and average total DP fluorescence intensity in the conditions depicted in C shows that shift of DP from cytoplasm to cell–cell border is largely absent in Pkp3 KD cells. Average total fluorescence intensity was normalized to represent 100% in each condition. The p values for control-to-Pkp3 KD comparisons are as follows (ANOVA, Bonferroni): 30 min cytoplasmic DP: ns, p > 0.05. All other cytoplasmic and cell–cell border DP measurements: p
    Figure Legend Snippet: Pkp3 mediates recruitment of soluble cytoplasmic DP to the sites of cell–cell contacts. (A) Western blot showing the difference in the presence of DP in cytoplasmic (saponin soluble) and membrane/cytoskeleton-bound (urea soluble) fractions in Pkp3 KD cells compared with control and Pkp2 KD cells. GAPDH and keratin 18 (K18) were used as loading controls for their respective fractions. (B) Western blot showing the levels of DP in the cytoplasmic (saponin soluble) cell fraction under conditions of calcium switch. GAPDH- normalized DP band intensity for each condition was determined using ImageJ. Ratio between soluble cytoplasmic DP in control and Pkp3 KD cells shows a rapid decrease in cytoplasmic DP in control cells. (C) Representative immunofluorescence images of DP appearing at cell borders after calcium switch from low to calcium concentration permissive for cell–cell junction formation, after the time indicated. “High Ca2+” represents cells that were switched overnight and are identical to steady-state conditions. White solid rectangles in the images to the left delineate areas enlarged at the right. Scale bar, 20 μm. (D) Ratio between the average fluorescence pixel intensities of cell–cell border and cytoplasmic DP and average total DP fluorescence intensity in the conditions depicted in C shows that shift of DP from cytoplasm to cell–cell border is largely absent in Pkp3 KD cells. Average total fluorescence intensity was normalized to represent 100% in each condition. The p values for control-to-Pkp3 KD comparisons are as follows (ANOVA, Bonferroni): 30 min cytoplasmic DP: ns, p > 0.05. All other cytoplasmic and cell–cell border DP measurements: p

    Techniques Used: Western Blot, Immunofluorescence, Concentration Assay, Fluorescence

    Activation of cAMP pathway reverses effects of Pkp3 ablation on desmosomal assembly and adhesion strength. (A) Immunofluorescence staining showing the effects on DP border localization of a PKC activator (PMA) and a RhoA inhibitor (C3) in Pkp2 KD and adenylyl cyclase activator (FSK) in Pkp3 KD SCC9 cells. Scale bar, 20 μm. (B) Immunofluorescence staining of 3D raft cultures after 6 d of differentiation shows recovery in DP distribution in Pkp3 KD rafts upon FSK treatment. Scale bar, 50 μm. (C) Immunofluorescence analysis of DP distribution in control and Pkp3-deficient SCC9 cells treated with vehicle, adrenergic agonist (ISO), or adrenergic antagonist (PROP). Scale bar, 20 μm. Note the recovery in ISO-treated Pkp3 KD and disruption of DP in PROP-treated control cells. (D) Cartoon depicting cAMP signaling with the activators and inhibitors used in this work. (E) Average fluorescence pixel intensities of DP at cell–cell borders of control and Pkp3 KD cells treated as specified in A (top, FSK) and C (bottom, ISO, PROP). Bars represent mean ± SEM. ns, p > 0.05, **0.001
    Figure Legend Snippet: Activation of cAMP pathway reverses effects of Pkp3 ablation on desmosomal assembly and adhesion strength. (A) Immunofluorescence staining showing the effects on DP border localization of a PKC activator (PMA) and a RhoA inhibitor (C3) in Pkp2 KD and adenylyl cyclase activator (FSK) in Pkp3 KD SCC9 cells. Scale bar, 20 μm. (B) Immunofluorescence staining of 3D raft cultures after 6 d of differentiation shows recovery in DP distribution in Pkp3 KD rafts upon FSK treatment. Scale bar, 50 μm. (C) Immunofluorescence analysis of DP distribution in control and Pkp3-deficient SCC9 cells treated with vehicle, adrenergic agonist (ISO), or adrenergic antagonist (PROP). Scale bar, 20 μm. Note the recovery in ISO-treated Pkp3 KD and disruption of DP in PROP-treated control cells. (D) Cartoon depicting cAMP signaling with the activators and inhibitors used in this work. (E) Average fluorescence pixel intensities of DP at cell–cell borders of control and Pkp3 KD cells treated as specified in A (top, FSK) and C (bottom, ISO, PROP). Bars represent mean ± SEM. ns, p > 0.05, **0.001

    Techniques Used: Activation Assay, Immunofluorescence, Staining, Fluorescence

    Pkp3 deficiency causes defects in cAMP-dependent E-cad maturation. (A) Immunofluorescence staining of E-cad in SCC9 (left) and HaCaT (right) cells shows aberrant cell–cell junction localization in steady-state high-calcium conditions in Pkp3KD cells compared with the control. (B) Immunofluorescence staining showing a defect in maturation of E-cad upon calcium switch at the sites of cell–cell contact in the Pkp3 KD SCC9 cells compared with control. (C) Immunofluorescence staining showing the recovery of E-cad at the borders of Pkp3 KD cells treated with forskolin. Conversely, control cells treated with propranolol mimic the immature appearance of E-cad junctions in a manner similar to the Pkp3 KD cells. Scale bars, 20 μm.
    Figure Legend Snippet: Pkp3 deficiency causes defects in cAMP-dependent E-cad maturation. (A) Immunofluorescence staining of E-cad in SCC9 (left) and HaCaT (right) cells shows aberrant cell–cell junction localization in steady-state high-calcium conditions in Pkp3KD cells compared with the control. (B) Immunofluorescence staining showing a defect in maturation of E-cad upon calcium switch at the sites of cell–cell contact in the Pkp3 KD SCC9 cells compared with control. (C) Immunofluorescence staining showing the recovery of E-cad at the borders of Pkp3 KD cells treated with forskolin. Conversely, control cells treated with propranolol mimic the immature appearance of E-cad junctions in a manner similar to the Pkp3 KD cells. Scale bars, 20 μm.

    Techniques Used: Immunofluorescence, Staining

    Pkp3 is required for efficient assembly of DP into desmosomes. (A) Immunofluorescence staining for DP representing the distinct patterns of its disruption in Pkp2, Pkp3, and Pkp2-3 double-KD SCC9 cells. Scale bar, 20 μm. Yellow dashed rectangles in top images delineate areas enlarged at the bottom. Yellow arrows point to the sites of cell–cell contacts; red arrows point to DP-containing nonvesicular desmosome precursors in the cytoplasm. Note the absence of cytoplasmic particles in Pkp3 KD and both particles and cell–cell border DP in double-KD cells. (B) Average fluorescence DP pixel intensity at the cell–cell borders and inside the cytoplasm, measured for ≥100 individual cells, showed decreased DP at cell–cell borders in Pkp3 KD cells and a corresponding increase in the cytoplasm. Error bars represent ± SEM. *** p
    Figure Legend Snippet: Pkp3 is required for efficient assembly of DP into desmosomes. (A) Immunofluorescence staining for DP representing the distinct patterns of its disruption in Pkp2, Pkp3, and Pkp2-3 double-KD SCC9 cells. Scale bar, 20 μm. Yellow dashed rectangles in top images delineate areas enlarged at the bottom. Yellow arrows point to the sites of cell–cell contacts; red arrows point to DP-containing nonvesicular desmosome precursors in the cytoplasm. Note the absence of cytoplasmic particles in Pkp3 KD and both particles and cell–cell border DP in double-KD cells. (B) Average fluorescence DP pixel intensity at the cell–cell borders and inside the cytoplasm, measured for ≥100 individual cells, showed decreased DP at cell–cell borders in Pkp3 KD cells and a corresponding increase in the cytoplasm. Error bars represent ± SEM. *** p

    Techniques Used: Immunofluorescence, Staining, Fluorescence

    Model for the role of Pkp3 in cell–cell junction formation. Left, activation of cAMP pathway upon cell–cell contact in high calcium leads to rapid recruitment of Pkp3-Rap1 complex to E-cad. At the same time, juxtamembrane DP coalesces at the sites of cell–cell contacts, requiring either Pkp2 or Pkp3, forming nascent desmosomes. Functional Pkp3-Rap1-E-cad complex drives adherens junction maturation by pulling the cells closer together and desmosome assembly by signaling the formation of cytoplasmic DP particles. On the other hand, Pkp3 acts as a spacer, preventing the aberrant coalescence of nascent desmosomes at the membrane. Pkp2 harnesses the activity of PKCα and RhoA to facilitate actin-dependent transport of cytoplasmic DP particles to the membrane. Right, model of steady-state adherens and desmosome junctions demonstrating mature cortical actin distribution and correctly sealed adherens junctions in the presence of Pkp3. In the absence of Pkp3 (not shown), Pkp3-Rap1-E-cad complex fails to assemble, leading to immature adherens junctions and failure of DP cytoplasmic particles to form. In addition, aberrant coalescence of nascent desmosomes occurs at the membrane.
    Figure Legend Snippet: Model for the role of Pkp3 in cell–cell junction formation. Left, activation of cAMP pathway upon cell–cell contact in high calcium leads to rapid recruitment of Pkp3-Rap1 complex to E-cad. At the same time, juxtamembrane DP coalesces at the sites of cell–cell contacts, requiring either Pkp2 or Pkp3, forming nascent desmosomes. Functional Pkp3-Rap1-E-cad complex drives adherens junction maturation by pulling the cells closer together and desmosome assembly by signaling the formation of cytoplasmic DP particles. On the other hand, Pkp3 acts as a spacer, preventing the aberrant coalescence of nascent desmosomes at the membrane. Pkp2 harnesses the activity of PKCα and RhoA to facilitate actin-dependent transport of cytoplasmic DP particles to the membrane. Right, model of steady-state adherens and desmosome junctions demonstrating mature cortical actin distribution and correctly sealed adherens junctions in the presence of Pkp3. In the absence of Pkp3 (not shown), Pkp3-Rap1-E-cad complex fails to assemble, leading to immature adherens junctions and failure of DP cytoplasmic particles to form. In addition, aberrant coalescence of nascent desmosomes occurs at the membrane.

    Techniques Used: Activation Assay, Functional Assay, Activity Assay

    Pkp3-Rap1 complex is required for maintaining physical interaction between Rap1 and E-cad. (A) PLA (red dots) showing which of the molecular pairs indicated are in close proximity (within 100-nm range) within cells. Scale bar, 20 μm. (B) Western blot showing the immunoprecipitation (left) and total (right) protein levels of Pkp3, Rap1, E-cad, and Pkp2 for indicated conditions (with GAPDH as 5% loading control; asterisk indicates the Rap1 band just above the IgG light chain). Note the loss of E-cad from Rap1 complexes in Pkp3 KD condition. (C) Immunofluorescence staining showing the cell–cell border localization of E-cad in relation to DP (top) and Pkp3 (bottom). Note the minimal overlap between DP and E-cad and a substantial overlap between E-cad and Pkp3. Scale bar, 20 μm. (D) Western blot showing the immunoprecipitation (left) and total (right) protein levels of E-cad, Pkp3, Rap1, and EPAC for indicated conditions. Note that E-cad loss does not interfere with the interaction between Pkp3, Rap1, and EPAC. (E) Western blot showing the immunoprecipitation (left) and total (right) protein levels of E-cad, Pkp3, and Rap1 for indicated conditions. Note that the EPAC inhibitor ESI-09 causes the loss of E-cad but not Pkp3 from the Rap1 complex.
    Figure Legend Snippet: Pkp3-Rap1 complex is required for maintaining physical interaction between Rap1 and E-cad. (A) PLA (red dots) showing which of the molecular pairs indicated are in close proximity (within 100-nm range) within cells. Scale bar, 20 μm. (B) Western blot showing the immunoprecipitation (left) and total (right) protein levels of Pkp3, Rap1, E-cad, and Pkp2 for indicated conditions (with GAPDH as 5% loading control; asterisk indicates the Rap1 band just above the IgG light chain). Note the loss of E-cad from Rap1 complexes in Pkp3 KD condition. (C) Immunofluorescence staining showing the cell–cell border localization of E-cad in relation to DP (top) and Pkp3 (bottom). Note the minimal overlap between DP and E-cad and a substantial overlap between E-cad and Pkp3. Scale bar, 20 μm. (D) Western blot showing the immunoprecipitation (left) and total (right) protein levels of E-cad, Pkp3, Rap1, and EPAC for indicated conditions. Note that E-cad loss does not interfere with the interaction between Pkp3, Rap1, and EPAC. (E) Western blot showing the immunoprecipitation (left) and total (right) protein levels of E-cad, Pkp3, and Rap1 for indicated conditions. Note that the EPAC inhibitor ESI-09 causes the loss of E-cad but not Pkp3 from the Rap1 complex.

    Techniques Used: Proximity Ligation Assay, Western Blot, Immunoprecipitation, Immunofluorescence, Staining

    8) Product Images from "The Rho kinases I and II regulate different aspects of myosin II activity"

    Article Title: The Rho kinases I and II regulate different aspects of myosin II activity

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200412043

    ROCK I but not ROCK II activity is essential for focal adhesion and stress fiber formation. (A) Fibroblast cultures were transfected with ROCK I, ROCK II, negative control, or a mixture of ROCK I and II siRNA duplexes. After maintenance culture for 2 d, cells were fixed and stained for paxillin and F-actin. Arrowheads indicate transfected cells. 2 d after transfection with siRNA duplexes, REF were incubated with Y-27632 (10 μM) in the presence of FCS. After 30 min, cells were fixed and stained for F-actin. In all cases, stress fibers were sensitive to Y-27632 addition. (B) Total cell lysates of REF prepared 2 d after transfection with ROCK I (I), ROCK II (II), or control (C) siRNA duplexes were analyzed by Western blotting for ROCK I, ROCK II, and actin. In each case, total populations were analyzed, containing some nontransfected cells. Results from cultures where 80–90% of cells were transfected are shown. (C) REF were cotransfected with myc-tagged full-length human ROCK I or bovine ROCK II cDNA and cy3-labeled ROCK I or II siRNA duplexes. 2 d after transfection, cells were fixed and stained for myc and F-actin. Either ROCK cDNA can restore a normal phenotype. Arrowheads indicate cotransfected cells. Bars, 50 μm.
    Figure Legend Snippet: ROCK I but not ROCK II activity is essential for focal adhesion and stress fiber formation. (A) Fibroblast cultures were transfected with ROCK I, ROCK II, negative control, or a mixture of ROCK I and II siRNA duplexes. After maintenance culture for 2 d, cells were fixed and stained for paxillin and F-actin. Arrowheads indicate transfected cells. 2 d after transfection with siRNA duplexes, REF were incubated with Y-27632 (10 μM) in the presence of FCS. After 30 min, cells were fixed and stained for F-actin. In all cases, stress fibers were sensitive to Y-27632 addition. (B) Total cell lysates of REF prepared 2 d after transfection with ROCK I (I), ROCK II (II), or control (C) siRNA duplexes were analyzed by Western blotting for ROCK I, ROCK II, and actin. In each case, total populations were analyzed, containing some nontransfected cells. Results from cultures where 80–90% of cells were transfected are shown. (C) REF were cotransfected with myc-tagged full-length human ROCK I or bovine ROCK II cDNA and cy3-labeled ROCK I or II siRNA duplexes. 2 d after transfection, cells were fixed and stained for myc and F-actin. Either ROCK cDNA can restore a normal phenotype. Arrowheads indicate cotransfected cells. Bars, 50 μm.

    Techniques Used: Activity Assay, Transfection, Negative Control, Staining, Incubation, Western Blot, Labeling

    9) Product Images from "Acute depletion of Tet1-dependent 5-hydroxymethylcytosine levels impairs LIF/Stat3 signaling and results in loss of embryonic stem cell identity"

    Article Title: Acute depletion of Tet1-dependent 5-hydroxymethylcytosine levels impairs LIF/Stat3 signaling and results in loss of embryonic stem cell identity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1253

    Depletion of Tet1 and 5hmC levels results in loss of mouse embryonic stem identity. ( A ) Oct4GiP mESCs were transfected with indicated siRNAs in normal ESC medium and cultured for 96 h. The percentage of differentiated cells was determined by measuring the percentage of GFP-negative cells using FACS at 96 h after transfection (** P
    Figure Legend Snippet: Depletion of Tet1 and 5hmC levels results in loss of mouse embryonic stem identity. ( A ) Oct4GiP mESCs were transfected with indicated siRNAs in normal ESC medium and cultured for 96 h. The percentage of differentiated cells was determined by measuring the percentage of GFP-negative cells using FACS at 96 h after transfection (** P

    Techniques Used: Transfection, Cell Culture, FACS

    10) Product Images from "Knockout mouse production assisted by Blm knockdown"

    Article Title: Knockout mouse production assisted by Blm knockdown

    Journal: The Journal of Reproduction and Development

    doi: 10.1262/jrd.2015-122

    Blm was knocked down by siRNAs. a) 20 nM of each Blm siRNA was transfected into KY1.1. At 48 h after transfection, the Blm mRNA level was measured by quantitative RT-PCR using two primer sets. b) The expression level of Blm protein was determined by western blot. Blm is indicated by an arrowhead. As a control for Blm knockdown, the Blm conditional knockdown ESC line, Blm tet/tet [ 14 ], was used. Tubulin was used as an internal control for protein content. Si (–), no siRNA; siC-L, siRNA Negative Control Low Duplex; siC-M, Medium Duplex; Tet, Tetracycline c. Blm was knocked down by a mixture of siBlm-2 and siBlm-3 in KY1.1. This protocol was used for the gene targeting experiments. Blm expression was determined by quantitative RT-PCR (upper panel) and western blot (lower panel). All data are presented as the mean ± SE.
    Figure Legend Snippet: Blm was knocked down by siRNAs. a) 20 nM of each Blm siRNA was transfected into KY1.1. At 48 h after transfection, the Blm mRNA level was measured by quantitative RT-PCR using two primer sets. b) The expression level of Blm protein was determined by western blot. Blm is indicated by an arrowhead. As a control for Blm knockdown, the Blm conditional knockdown ESC line, Blm tet/tet [ 14 ], was used. Tubulin was used as an internal control for protein content. Si (–), no siRNA; siC-L, siRNA Negative Control Low Duplex; siC-M, Medium Duplex; Tet, Tetracycline c. Blm was knocked down by a mixture of siBlm-2 and siBlm-3 in KY1.1. This protocol was used for the gene targeting experiments. Blm expression was determined by quantitative RT-PCR (upper panel) and western blot (lower panel). All data are presented as the mean ± SE.

    Techniques Used: Transfection, Quantitative RT-PCR, Expressing, Western Blot, Negative Control

    11) Product Images from "Regulation of α2B-Adrenergic Receptor Cell Surface Transport by GGA1 and GGA2"

    Article Title: Regulation of α2B-Adrenergic Receptor Cell Surface Transport by GGA1 and GGA2

    Journal: Scientific Reports

    doi: 10.1038/srep37921

    Effect of GGA1 and GGA2 depletion on the dendritic expression of α 2B -AR in primary cortical neurons. ( A ) Effect of GGA1 knockdown on α 2B -AR expression in the dendrites of primary cortical neurons. The cortical neurons were transfected with α 2B -AR-GFP together with GGA1 siRNA at DIV 5. Two days after transfection, the neurons were stained with antibodies against GGA1. The distribution of α 2B -AR was visualized by confocal microscopy. ( B ) Effect of GGA2 knockdown on the dendritic expression of α 2B -AR. The data shown are representative images in at least 4 individual experiments. Arrows indicate the expression of GGA1 or GGA2. Scale bars, 20 μm. ( C ) Quantitative data shown in ( A , B ) (n = 17). α 2B -AR expression in the dendrites was determined by measuring the GFP signal. * p
    Figure Legend Snippet: Effect of GGA1 and GGA2 depletion on the dendritic expression of α 2B -AR in primary cortical neurons. ( A ) Effect of GGA1 knockdown on α 2B -AR expression in the dendrites of primary cortical neurons. The cortical neurons were transfected with α 2B -AR-GFP together with GGA1 siRNA at DIV 5. Two days after transfection, the neurons were stained with antibodies against GGA1. The distribution of α 2B -AR was visualized by confocal microscopy. ( B ) Effect of GGA2 knockdown on the dendritic expression of α 2B -AR. The data shown are representative images in at least 4 individual experiments. Arrows indicate the expression of GGA1 or GGA2. Scale bars, 20 μm. ( C ) Quantitative data shown in ( A , B ) (n = 17). α 2B -AR expression in the dendrites was determined by measuring the GFP signal. * p

    Techniques Used: Expressing, Transfection, Staining, Confocal Microscopy

    Inhibition of cell surface expression of α 2B -AR by siRNA-mediated depletion of GGA1 and GGA2. ( A ) siRNA-mediated depletion of GGA1 and GGA2 in HEK293 cells. ( B ) Effect of siRNA-mediated knockdown of GGA1 and GGA2 on the cell surface expression of α 2B -AR. HEK293 cells inducibly expressing α 2B -AR were transfected with control siRNA or siRNA targeting GGA1 and GGA2 and incubated with doxycycline as described in legends of Fig. 1B . The average specific binding of [ 3 H]-RX821002 from cells without siRNA transfection and treated with doxycycline for 28 h was 34,423 ± 563 cpm per well. ( C ) Effect of combination knockdown of GGA1, GGA2 and GGA3 on the cell surface expression of α 2B -AR in HEK293 cells. ( D ) Effect of knockdown of GGA1, GGA2 and GGA3 on the Golgi structure. HEK293 cells were transfected with control or GGA siRNA for 48 h and then stained with antibodies against GM130 (1:200 dilution) and p230 (1:100 dilution) overnight. Scale bar, 10 μm. ( E ) Effect of GGA1 and GGA2 knockdown on total α 2B -AR expression. HEK293 cells inducibly expressing α 2B -AR were transfected with control or GGA shRNA or siRNA for 24 h and incubated with doxycycline (40 ng/ml) for another 24 h. The overall α 2B -AR expression was measured by flow cytometry following staining with HA antibodies in permeabilized cells (n = 3). ( F ) Effect of GGA1 and GGA2 knockdown on the internalization of α 2B -AR. HEK293 cells stably expressing α 2B -AR were transfected with arrestin-3 and control or GGA shRNA and incubated with doxycycline as described above. The cells were then stimulated with epinephrine (100 μM) for 10, 20 and 30 min (n = 3). The cell surface expression of α 2B -AR was determined by intact cell ligand binding using [ 3 H]-RX821002. The data are presented as the mean ± S.E. of at least three individual experiments in ( B , C , E , F ). * p
    Figure Legend Snippet: Inhibition of cell surface expression of α 2B -AR by siRNA-mediated depletion of GGA1 and GGA2. ( A ) siRNA-mediated depletion of GGA1 and GGA2 in HEK293 cells. ( B ) Effect of siRNA-mediated knockdown of GGA1 and GGA2 on the cell surface expression of α 2B -AR. HEK293 cells inducibly expressing α 2B -AR were transfected with control siRNA or siRNA targeting GGA1 and GGA2 and incubated with doxycycline as described in legends of Fig. 1B . The average specific binding of [ 3 H]-RX821002 from cells without siRNA transfection and treated with doxycycline for 28 h was 34,423 ± 563 cpm per well. ( C ) Effect of combination knockdown of GGA1, GGA2 and GGA3 on the cell surface expression of α 2B -AR in HEK293 cells. ( D ) Effect of knockdown of GGA1, GGA2 and GGA3 on the Golgi structure. HEK293 cells were transfected with control or GGA siRNA for 48 h and then stained with antibodies against GM130 (1:200 dilution) and p230 (1:100 dilution) overnight. Scale bar, 10 μm. ( E ) Effect of GGA1 and GGA2 knockdown on total α 2B -AR expression. HEK293 cells inducibly expressing α 2B -AR were transfected with control or GGA shRNA or siRNA for 24 h and incubated with doxycycline (40 ng/ml) for another 24 h. The overall α 2B -AR expression was measured by flow cytometry following staining with HA antibodies in permeabilized cells (n = 3). ( F ) Effect of GGA1 and GGA2 knockdown on the internalization of α 2B -AR. HEK293 cells stably expressing α 2B -AR were transfected with arrestin-3 and control or GGA shRNA and incubated with doxycycline as described above. The cells were then stimulated with epinephrine (100 μM) for 10, 20 and 30 min (n = 3). The cell surface expression of α 2B -AR was determined by intact cell ligand binding using [ 3 H]-RX821002. The data are presented as the mean ± S.E. of at least three individual experiments in ( B , C , E , F ). * p

    Techniques Used: Inhibition, Expressing, Transfection, Incubation, Binding Assay, Staining, shRNA, Flow Cytometry, Cytometry, Stable Transfection, Ligand Binding Assay

    Interaction of α 2B -AR with GGA1 and GGA2. ( A ) Interaction of α 2B -AR with GGA1 and GGA2 in co-immunoprecipitation assays. HEK293 cells stably expressing HA-α 2B -AR were transfected with control vector or myc-tagged GGA1 and GGA2. The receptors were immunoprecipitated with α 2B -AR antibodies. The amounts of GGA1 and GGA2 (upper panel) and α 2B -AR (lower panel) were determined by immunoblotting using myc and α 2B -AR antibodies, respectively. Lysate - 1% of total input. Similar results were obtained in three experiments. ( B ) Sequences of the ICL1, ICL2, ICL3 and C-terminus (CT) of α 2B -AR (upper panel) and Coomassie blue staining of purified GST fusion proteins (low panel). The calculated molecular weights of GST and the ICL1, ICL2, ICL3, and CT GST fusion proteins are 27,898, 27,422, 28,070, 43,779 and 29,348 daltons, respectively. ( C ) Interaction of different intracellular domains of α 2B -AR with GGA1 and GGA2. Myc-tagged GGA1 and GGA2 were expressed in HEK293 cells and total cell homogenates were incubated with GST fusion proteins. Bound GGAs were revealed by immunoblotting using anti-myc antibodies. ( D ) Purified His-tagged GGA1 and GGA2. The molecular weight (MW) markers (KDa) are indicated on the left. ( E ) Direct interaction of the α 2B -AR ICL3 with GGA1 and GGA2. Purified His-tagged GGA1 and GGA2 were incubated with GST-ICL3 fusion proteins and bound GGAs were detected by immunoblotting using anti-His antibodies. Similar results were obtained in at least three separate experiments. Lysate −5% of total input. Similar results were obtained in at least 3 experiments.
    Figure Legend Snippet: Interaction of α 2B -AR with GGA1 and GGA2. ( A ) Interaction of α 2B -AR with GGA1 and GGA2 in co-immunoprecipitation assays. HEK293 cells stably expressing HA-α 2B -AR were transfected with control vector or myc-tagged GGA1 and GGA2. The receptors were immunoprecipitated with α 2B -AR antibodies. The amounts of GGA1 and GGA2 (upper panel) and α 2B -AR (lower panel) were determined by immunoblotting using myc and α 2B -AR antibodies, respectively. Lysate - 1% of total input. Similar results were obtained in three experiments. ( B ) Sequences of the ICL1, ICL2, ICL3 and C-terminus (CT) of α 2B -AR (upper panel) and Coomassie blue staining of purified GST fusion proteins (low panel). The calculated molecular weights of GST and the ICL1, ICL2, ICL3, and CT GST fusion proteins are 27,898, 27,422, 28,070, 43,779 and 29,348 daltons, respectively. ( C ) Interaction of different intracellular domains of α 2B -AR with GGA1 and GGA2. Myc-tagged GGA1 and GGA2 were expressed in HEK293 cells and total cell homogenates were incubated with GST fusion proteins. Bound GGAs were revealed by immunoblotting using anti-myc antibodies. ( D ) Purified His-tagged GGA1 and GGA2. The molecular weight (MW) markers (KDa) are indicated on the left. ( E ) Direct interaction of the α 2B -AR ICL3 with GGA1 and GGA2. Purified His-tagged GGA1 and GGA2 were incubated with GST-ICL3 fusion proteins and bound GGAs were detected by immunoblotting using anti-His antibodies. Similar results were obtained in at least three separate experiments. Lysate −5% of total input. Similar results were obtained in at least 3 experiments.

    Techniques Used: Immunoprecipitation, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Staining, Purification, Incubation, Molecular Weight

    Effect of depleting GGA1 and GGA2 on α 2B -AR-mediated signaling. ( A ) HEK293 cells were transfected with control shRNA or individual GGA shRNA for 36 h. After starvation for 3 h, the cells were stimulated with UK14304 at the concentration of 1 μM for 5 min at 37 °C. ERK1/2 activation was determined by Western blot analysis using phospho-specific ERK1/2 antibodies. Upper panel is a representative blot of ERK1/2 activation and lower panel shows total ERK1/2 expression. ( B ) Quantitative data shown in A). The data shown are percentages of the mean value obtained from cells transfected with control shRNA and are presented as the mean ± S.E. of at least three experiments. * p
    Figure Legend Snippet: Effect of depleting GGA1 and GGA2 on α 2B -AR-mediated signaling. ( A ) HEK293 cells were transfected with control shRNA or individual GGA shRNA for 36 h. After starvation for 3 h, the cells were stimulated with UK14304 at the concentration of 1 μM for 5 min at 37 °C. ERK1/2 activation was determined by Western blot analysis using phospho-specific ERK1/2 antibodies. Upper panel is a representative blot of ERK1/2 activation and lower panel shows total ERK1/2 expression. ( B ) Quantitative data shown in A). The data shown are percentages of the mean value obtained from cells transfected with control shRNA and are presented as the mean ± S.E. of at least three experiments. * p

    Techniques Used: Transfection, shRNA, Concentration Assay, Activation Assay, Western Blot, Expressing

    Identification of the α 2B -AR-binding sites in GGA1 and GGA2. ( A ) Subcellular distribution of GGA1 and GGA2 and their domains revealed by confocal microscopy. HeLa cells were transfected with GFP-tagged GGA1, GGA2 or individual domains for 24 h. Similar results were obtained in at least three separate experiments. Scale bar, 10 μm. ( B ) Interaction of different domains of GGA1 and GGA2 with GST-ICL3. Amino acid sequence analyses showed that the identities of the VHS, GAT, hinge and GAE domains between GGA1 and GGA2 are approximately 65, 61, 26 and 55%, respectively. The GFP-tagged VHS, GAT, hinge and GAE domains of GGA1 and GGA2 were expressed in HEK293 cells. Total cell lysates were incubated with GST-ICL3 fusion proteins. Bound GGA domains were revealed by immunoblotting using GFP antibodies. Total cell lysates expressing GFP alone were used as a control. Lysate – 5% of total input. Similar results were obtained in at least three different experiments.
    Figure Legend Snippet: Identification of the α 2B -AR-binding sites in GGA1 and GGA2. ( A ) Subcellular distribution of GGA1 and GGA2 and their domains revealed by confocal microscopy. HeLa cells were transfected with GFP-tagged GGA1, GGA2 or individual domains for 24 h. Similar results were obtained in at least three separate experiments. Scale bar, 10 μm. ( B ) Interaction of different domains of GGA1 and GGA2 with GST-ICL3. Amino acid sequence analyses showed that the identities of the VHS, GAT, hinge and GAE domains between GGA1 and GGA2 are approximately 65, 61, 26 and 55%, respectively. The GFP-tagged VHS, GAT, hinge and GAE domains of GGA1 and GGA2 were expressed in HEK293 cells. Total cell lysates were incubated with GST-ICL3 fusion proteins. Bound GGA domains were revealed by immunoblotting using GFP antibodies. Total cell lysates expressing GFP alone were used as a control. Lysate – 5% of total input. Similar results were obtained in at least three different experiments.

    Techniques Used: Binding Assay, Confocal Microscopy, Transfection, Sequencing, Incubation, Expressing

    Inhibition of cell surface expression of α 2B -AR by shRNA-mediated knockdown of GGA1 and GGA2. ( A ) shRNA-mediated depletion of GGA1 and GGA2 in HEK293 cells. The expression of GGAs was measured by immunoblotting using isoform-specific antibodies. ( B ) Effect of shRNA-mediated knockdown of GGA1 and GGA2 on the cell surface expression of α 2B -AR. HEK293 cells inducibly expressing α 2B -AR were transfected with control or GGA shRNA and then incubated with doxycycline at the concentration of 40 ng/ml for different time periods (0, 4, 8, 12, 16, 20, 24 and 28 h). The cell surface expression of α 2B -AR was determined by intact cell ligand binding using [ 3 H]-RX821002. The data shown are percentages of specific binding obtained from cells transfected with control shRNA and treated with doxycycline for 28 h, in which the mean value of specific [ 3 H]-RX821002 binding was 35,642 ± 985 cpm per well (n = 4) and presented as the mean ± S.E. of at least three individual experiments. * p
    Figure Legend Snippet: Inhibition of cell surface expression of α 2B -AR by shRNA-mediated knockdown of GGA1 and GGA2. ( A ) shRNA-mediated depletion of GGA1 and GGA2 in HEK293 cells. The expression of GGAs was measured by immunoblotting using isoform-specific antibodies. ( B ) Effect of shRNA-mediated knockdown of GGA1 and GGA2 on the cell surface expression of α 2B -AR. HEK293 cells inducibly expressing α 2B -AR were transfected with control or GGA shRNA and then incubated with doxycycline at the concentration of 40 ng/ml for different time periods (0, 4, 8, 12, 16, 20, 24 and 28 h). The cell surface expression of α 2B -AR was determined by intact cell ligand binding using [ 3 H]-RX821002. The data shown are percentages of specific binding obtained from cells transfected with control shRNA and treated with doxycycline for 28 h, in which the mean value of specific [ 3 H]-RX821002 binding was 35,642 ± 985 cpm per well (n = 4) and presented as the mean ± S.E. of at least three individual experiments. * p

    Techniques Used: Inhibition, Expressing, shRNA, Transfection, Incubation, Concentration Assay, Ligand Binding Assay, Binding Assay

    Identification of the GGA1- and GGA2-binding sites in the α 2B -AR ICL3 by progressive deletion. ( A ) Interaction of different ICL3 fragments with the GGA1 hinge and the GGA2 GAE domains. Each ICL3 fragment was generated as GST fusion proteins. The GGA1 hinge and the GGA2 GAE domains were generated as GFP fusion proteins. Their interactions were determined in GST fusion protein pulldown assays. Bound GGA domains were revealed by immunoblotting using GFP antibodies. Bottom panel shows Coomassie blue staining of purified GST fusion proteins. Similar results were obtained in at least three different experiments. The blots from two gels that were run under the same experimental conditions were combined to show the interaction of the GGA1 hinge with different ICL3 domains (upper panel). ( B ) A summary of progressive deletion to identify the GGA1- and GGA2-binding domains in the α 2B -AR ICL3 as shown in ( A ). +Interacting with individual GGA domains; −, not interacting with GGA. ( C ) A diagram showing differential interactions between α 2B -AR and three GGAs. The GGA1 hinge and the GGA2 GAE domains bind to two subdomains of the α 2B -AR ICL3 as revealed in the current studies, whereas the GGA3 VHS domain interacts with the α 2B -AR ICL3, specifically the 3R motif, as demonstrated in our previous studies 47 .
    Figure Legend Snippet: Identification of the GGA1- and GGA2-binding sites in the α 2B -AR ICL3 by progressive deletion. ( A ) Interaction of different ICL3 fragments with the GGA1 hinge and the GGA2 GAE domains. Each ICL3 fragment was generated as GST fusion proteins. The GGA1 hinge and the GGA2 GAE domains were generated as GFP fusion proteins. Their interactions were determined in GST fusion protein pulldown assays. Bound GGA domains were revealed by immunoblotting using GFP antibodies. Bottom panel shows Coomassie blue staining of purified GST fusion proteins. Similar results were obtained in at least three different experiments. The blots from two gels that were run under the same experimental conditions were combined to show the interaction of the GGA1 hinge with different ICL3 domains (upper panel). ( B ) A summary of progressive deletion to identify the GGA1- and GGA2-binding domains in the α 2B -AR ICL3 as shown in ( A ). +Interacting with individual GGA domains; −, not interacting with GGA. ( C ) A diagram showing differential interactions between α 2B -AR and three GGAs. The GGA1 hinge and the GGA2 GAE domains bind to two subdomains of the α 2B -AR ICL3 as revealed in the current studies, whereas the GGA3 VHS domain interacts with the α 2B -AR ICL3, specifically the 3R motif, as demonstrated in our previous studies 47 .

    Techniques Used: Binding Assay, Generated, Staining, Purification

    12) Product Images from "The mitochondrial outer-membrane location of the EXD2 exonuclease contradicts its direct role in nuclear DNA repair"

    Article Title: The mitochondrial outer-membrane location of the EXD2 exonuclease contradicts its direct role in nuclear DNA repair

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-23690-y

    EXD2 is accessible to added antibody in immunofluorescence without mitochondrial lysis. Immunofluorescent detection following paraformaldehyde fixation requires mitochondrial lysis using for example Triton X100. In the absence of this lysis step mitochondrial matrix proteins and for example mtDNA are not detectable. Thus (panel a) results shows that in the absence of TX100 lysis, EXD2 is detected while neither MRPL12 nor mtDNA are detected by IF. With TX100 lysis, all three are detected. A similar experiment (panel b) shows that both Tomm20 and EXD2, but not mtDNA are detected in the absence of TX100 lysis. A high resolution 20 × 30 µM subsection of a cell using the EXD2 and MRPL12 antibodies illustrates that the EXD2 signal is often enveloping the MRPL12 signal (panel c: some examples are indicated by a white arrow in the merged image), further illustrating EXD2 its outer-membrane localization.
    Figure Legend Snippet: EXD2 is accessible to added antibody in immunofluorescence without mitochondrial lysis. Immunofluorescent detection following paraformaldehyde fixation requires mitochondrial lysis using for example Triton X100. In the absence of this lysis step mitochondrial matrix proteins and for example mtDNA are not detectable. Thus (panel a) results shows that in the absence of TX100 lysis, EXD2 is detected while neither MRPL12 nor mtDNA are detected by IF. With TX100 lysis, all three are detected. A similar experiment (panel b) shows that both Tomm20 and EXD2, but not mtDNA are detected in the absence of TX100 lysis. A high resolution 20 × 30 µM subsection of a cell using the EXD2 and MRPL12 antibodies illustrates that the EXD2 signal is often enveloping the MRPL12 signal (panel c: some examples are indicated by a white arrow in the merged image), further illustrating EXD2 its outer-membrane localization.

    Techniques Used: Immunofluorescence, Lysis

    SiRNA mediated knockdown of EXD2 in U2OS cells results in a more widely spread mitochondrial network. In order to understand possible effects of EXD2 knockdown on mitochondrial network behaviour we labelled cells with EdU to identify cells in S-phase, in order to get a handle on mosaicism resulting from cell-cycle mediated effects on mitochondrial network dynamics (see main text). For this, cells were incubated for 30 min with EdU following transfection and 48–60 hrs incubation with either a pool of non-targeting control siRNAs (Csi) or a pool of three EXD2 stealth siRNAs (EXD2si3x). Following labelling for EdU incorporation (green, gr), cells were further labelled with antibodies for Tomm20 (red, r) and EXD2 (white). Control siRNA treated cells show frequent and strong perinuclear clustering of the mitochondrial network in particular in EdU positive cells. In contrast, the EXD2si3x typically show a more distributed and sometimes more hyperfused network. Two fields of view are shown for each condition.
    Figure Legend Snippet: SiRNA mediated knockdown of EXD2 in U2OS cells results in a more widely spread mitochondrial network. In order to understand possible effects of EXD2 knockdown on mitochondrial network behaviour we labelled cells with EdU to identify cells in S-phase, in order to get a handle on mosaicism resulting from cell-cycle mediated effects on mitochondrial network dynamics (see main text). For this, cells were incubated for 30 min with EdU following transfection and 48–60 hrs incubation with either a pool of non-targeting control siRNAs (Csi) or a pool of three EXD2 stealth siRNAs (EXD2si3x). Following labelling for EdU incorporation (green, gr), cells were further labelled with antibodies for Tomm20 (red, r) and EXD2 (white). Control siRNA treated cells show frequent and strong perinuclear clustering of the mitochondrial network in particular in EdU positive cells. In contrast, the EXD2si3x typically show a more distributed and sometimes more hyperfused network. Two fields of view are shown for each condition.

    Techniques Used: Incubation, Transfection

    EXD2 is a mitochondrial outer-membrane associated protein. U2OS cell crude mitochondria isolated by differential centrifugation and pure nuclei isolated on iodixanol gradients were tested for EXD2 abundance in control siRNA and EXD2 siRNA treated cells (panel a1). Results show that the vast majority of EXD2, similar to mitochondrial marker proteins mtSSB and porin are found in the mitochondrial fraction and not in the nuclear fraction, in which the nuclear marker nucleophosmin is identified. SiRNA treatment confirms the identity of the full length (fl) ~70 kDa EXD2 protein and several lower abundant species of 30 kDa and higher molecular weight. Knockdown with either the combined pool of three commercial siRNAs or each individual siRNA show similar knockdown efficiencies in total cell lysates of U2OS cells on Western blots (panel a2). At the same time, several lower molecular weight EXD2 species are also identified and all appear to be equally sensitive to each individual siRNA suggesting they might be breakdown or processed EXD2 forms. Protease protection demonstrates mitochondrial EXD2 is mostly found in the mitochondrial outer-membrane (panel b). Mitochondria and digitonin-derived mitoplasts from HEK293 cells were treated either with Proteinase K (ProtK) alone or with ProtK and Triton-X100 (TX100) to lyse the inner and outer membrane. Results show that whereas TFAM, an mtDNA associated protein, is protected from ProtK in the absence of TX100, EXD2 is not, suggesting and outer-membrane localization. *Indicates a remnant signal from the probing with an antibody against a different mitochondrial candidate protein not relevant for this paper. A Na 2 CO 3 extract of crude mitochondria from HEK293 cells (panel c) shows that similar to cytochrome c oxidase subunit I (an integral membrane protein), full length EXD2 is found predominantly in the pellet (membrane) fraction, whereas the majority of HSP60 is found in the supernatant (non-membrane) fraction. For each panel (except panel b) cropped images show the results of incubations with subsequent antibodies on the same blots, indicated by dividing lines (see Supplementary info for full blot images).
    Figure Legend Snippet: EXD2 is a mitochondrial outer-membrane associated protein. U2OS cell crude mitochondria isolated by differential centrifugation and pure nuclei isolated on iodixanol gradients were tested for EXD2 abundance in control siRNA and EXD2 siRNA treated cells (panel a1). Results show that the vast majority of EXD2, similar to mitochondrial marker proteins mtSSB and porin are found in the mitochondrial fraction and not in the nuclear fraction, in which the nuclear marker nucleophosmin is identified. SiRNA treatment confirms the identity of the full length (fl) ~70 kDa EXD2 protein and several lower abundant species of 30 kDa and higher molecular weight. Knockdown with either the combined pool of three commercial siRNAs or each individual siRNA show similar knockdown efficiencies in total cell lysates of U2OS cells on Western blots (panel a2). At the same time, several lower molecular weight EXD2 species are also identified and all appear to be equally sensitive to each individual siRNA suggesting they might be breakdown or processed EXD2 forms. Protease protection demonstrates mitochondrial EXD2 is mostly found in the mitochondrial outer-membrane (panel b). Mitochondria and digitonin-derived mitoplasts from HEK293 cells were treated either with Proteinase K (ProtK) alone or with ProtK and Triton-X100 (TX100) to lyse the inner and outer membrane. Results show that whereas TFAM, an mtDNA associated protein, is protected from ProtK in the absence of TX100, EXD2 is not, suggesting and outer-membrane localization. *Indicates a remnant signal from the probing with an antibody against a different mitochondrial candidate protein not relevant for this paper. A Na 2 CO 3 extract of crude mitochondria from HEK293 cells (panel c) shows that similar to cytochrome c oxidase subunit I (an integral membrane protein), full length EXD2 is found predominantly in the pellet (membrane) fraction, whereas the majority of HSP60 is found in the supernatant (non-membrane) fraction. For each panel (except panel b) cropped images show the results of incubations with subsequent antibodies on the same blots, indicated by dividing lines (see Supplementary info for full blot images).

    Techniques Used: Isolation, Centrifugation, Marker, Molecular Weight, Western Blot, Derivative Assay

    Nuclear DNA-damage does not relocate EXD2 to the nucleus. (panel a) HeLa cells were treated with 13 Gy X-irradiation to induce nuclear DNA damage and allowed to recover for 30 minutes. Results show that H2AX phosphorylation is considerably increased in the nucleus following this damage whereas EXD2 retains its predominant mitochondrial localization similar to its location in control cells. (panel b) U2OS cells were treated with 250 µg/ml Zeocin for 1 hr and allowed to recover for 1 hr after replacing the Zeocin-containing medium with regular medium. Cells were then processed for IF detection of γH2AX and EXD2 and show a similar result as the X-irradiation in HeLa cells.
    Figure Legend Snippet: Nuclear DNA-damage does not relocate EXD2 to the nucleus. (panel a) HeLa cells were treated with 13 Gy X-irradiation to induce nuclear DNA damage and allowed to recover for 30 minutes. Results show that H2AX phosphorylation is considerably increased in the nucleus following this damage whereas EXD2 retains its predominant mitochondrial localization similar to its location in control cells. (panel b) U2OS cells were treated with 250 µg/ml Zeocin for 1 hr and allowed to recover for 1 hr after replacing the Zeocin-containing medium with regular medium. Cells were then processed for IF detection of γH2AX and EXD2 and show a similar result as the X-irradiation in HeLa cells.

    Techniques Used: Irradiation

    ). Overexpressed full length EXD2 showed no evidence of either nuclear or intermediate filament localization. Please note that for this Figure images have been selected deliberately to best illustrate the mitochondrial localization of EXD2. Image views have thus been chosen showing cells with a clear and extended mitochondrial network, while trying to avoid cells with a condensed/collapsed mitochondrial network.
    Figure Legend Snippet: ). Overexpressed full length EXD2 showed no evidence of either nuclear or intermediate filament localization. Please note that for this Figure images have been selected deliberately to best illustrate the mitochondrial localization of EXD2. Image views have thus been chosen showing cells with a clear and extended mitochondrial network, while trying to avoid cells with a condensed/collapsed mitochondrial network.

    Techniques Used:

    13) Product Images from "Plakophilin 3 mediates Rap1-dependent desmosome assembly and adherens junction maturation"

    Article Title: Plakophilin 3 mediates Rap1-dependent desmosome assembly and adherens junction maturation

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E14-05-0968

    Pkp3 ablation disrupts desmosomes. (A) Western blot showing no change in the levels of indicated desmosomal and adherens junction molecules in SCC9 cells in Pkp2 and 3 siRNA knockdown. (B) Immunofluorescence showing punctate Pkp2, Dsc2, Dsg2, and Pg staining at the sites of cell–cell contacts in Pkp3 KD SCC9 cells compared with control. Scale bar, 20 μm. (C) Electron micrographs demonstrating changes in size (yellow arrows, large, single desmosomes) and morphology (red arrows, large, tandem desmosomes) of desmosomes in Pkp3 KD as compared with the control SCC9 cells. Scale bar, 1 μm, 100 nm (enlargements). (D) Scatter plots showing an increase in length (top; triangles) and width (bottom; diamonds) of individual desmosomes observed by electron microscopy in control (blue) and Pkp3 KD (red). Horizontal lines represent mean ± SEM. *** p
    Figure Legend Snippet: Pkp3 ablation disrupts desmosomes. (A) Western blot showing no change in the levels of indicated desmosomal and adherens junction molecules in SCC9 cells in Pkp2 and 3 siRNA knockdown. (B) Immunofluorescence showing punctate Pkp2, Dsc2, Dsg2, and Pg staining at the sites of cell–cell contacts in Pkp3 KD SCC9 cells compared with control. Scale bar, 20 μm. (C) Electron micrographs demonstrating changes in size (yellow arrows, large, single desmosomes) and morphology (red arrows, large, tandem desmosomes) of desmosomes in Pkp3 KD as compared with the control SCC9 cells. Scale bar, 1 μm, 100 nm (enlargements). (D) Scatter plots showing an increase in length (top; triangles) and width (bottom; diamonds) of individual desmosomes observed by electron microscopy in control (blue) and Pkp3 KD (red). Horizontal lines represent mean ± SEM. *** p

    Techniques Used: Western Blot, Immunofluorescence, Staining, Electron Microscopy

    Pkp3 mediates recruitment of soluble cytoplasmic DP to the sites of cell–cell contacts. (A) Western blot showing the difference in the presence of DP in cytoplasmic (saponin soluble) and membrane/cytoskeleton-bound (urea soluble) fractions in Pkp3 KD cells compared with control and Pkp2 KD cells. GAPDH and keratin 18 (K18) were used as loading controls for their respective fractions. (B) Western blot showing the levels of DP in the cytoplasmic (saponin soluble) cell fraction under conditions of calcium switch. GAPDH- normalized DP band intensity for each condition was determined using ImageJ. Ratio between soluble cytoplasmic DP in control and Pkp3 KD cells shows a rapid decrease in cytoplasmic DP in control cells. (C) Representative immunofluorescence images of DP appearing at cell borders after calcium switch from low to calcium concentration permissive for cell–cell junction formation, after the time indicated. “High Ca2+” represents cells that were switched overnight and are identical to steady-state conditions. White solid rectangles in the images to the left delineate areas enlarged at the right. Scale bar, 20 μm. (D) Ratio between the average fluorescence pixel intensities of cell–cell border and cytoplasmic DP and average total DP fluorescence intensity in the conditions depicted in C shows that shift of DP from cytoplasm to cell–cell border is largely absent in Pkp3 KD cells. Average total fluorescence intensity was normalized to represent 100% in each condition. The p values for control-to-Pkp3 KD comparisons are as follows (ANOVA, Bonferroni): 30 min cytoplasmic DP: ns, p > 0.05. All other cytoplasmic and cell–cell border DP measurements: p
    Figure Legend Snippet: Pkp3 mediates recruitment of soluble cytoplasmic DP to the sites of cell–cell contacts. (A) Western blot showing the difference in the presence of DP in cytoplasmic (saponin soluble) and membrane/cytoskeleton-bound (urea soluble) fractions in Pkp3 KD cells compared with control and Pkp2 KD cells. GAPDH and keratin 18 (K18) were used as loading controls for their respective fractions. (B) Western blot showing the levels of DP in the cytoplasmic (saponin soluble) cell fraction under conditions of calcium switch. GAPDH- normalized DP band intensity for each condition was determined using ImageJ. Ratio between soluble cytoplasmic DP in control and Pkp3 KD cells shows a rapid decrease in cytoplasmic DP in control cells. (C) Representative immunofluorescence images of DP appearing at cell borders after calcium switch from low to calcium concentration permissive for cell–cell junction formation, after the time indicated. “High Ca2+” represents cells that were switched overnight and are identical to steady-state conditions. White solid rectangles in the images to the left delineate areas enlarged at the right. Scale bar, 20 μm. (D) Ratio between the average fluorescence pixel intensities of cell–cell border and cytoplasmic DP and average total DP fluorescence intensity in the conditions depicted in C shows that shift of DP from cytoplasm to cell–cell border is largely absent in Pkp3 KD cells. Average total fluorescence intensity was normalized to represent 100% in each condition. The p values for control-to-Pkp3 KD comparisons are as follows (ANOVA, Bonferroni): 30 min cytoplasmic DP: ns, p > 0.05. All other cytoplasmic and cell–cell border DP measurements: p

    Techniques Used: Western Blot, Immunofluorescence, Concentration Assay, Fluorescence

    Activation of cAMP pathway reverses effects of Pkp3 ablation on desmosomal assembly and adhesion strength. (A) Immunofluorescence staining showing the effects on DP border localization of a PKC activator (PMA) and a RhoA inhibitor (C3) in Pkp2 KD and adenylyl cyclase activator (FSK) in Pkp3 KD SCC9 cells. Scale bar, 20 μm. (B) Immunofluorescence staining of 3D raft cultures after 6 d of differentiation shows recovery in DP distribution in Pkp3 KD rafts upon FSK treatment. Scale bar, 50 μm. (C) Immunofluorescence analysis of DP distribution in control and Pkp3-deficient SCC9 cells treated with vehicle, adrenergic agonist (ISO), or adrenergic antagonist (PROP). Scale bar, 20 μm. Note the recovery in ISO-treated Pkp3 KD and disruption of DP in PROP-treated control cells. (D) Cartoon depicting cAMP signaling with the activators and inhibitors used in this work. (E) Average fluorescence pixel intensities of DP at cell–cell borders of control and Pkp3 KD cells treated as specified in A (top, FSK) and C (bottom, ISO, PROP). Bars represent mean ± SEM. ns, p > 0.05, **0.001
    Figure Legend Snippet: Activation of cAMP pathway reverses effects of Pkp3 ablation on desmosomal assembly and adhesion strength. (A) Immunofluorescence staining showing the effects on DP border localization of a PKC activator (PMA) and a RhoA inhibitor (C3) in Pkp2 KD and adenylyl cyclase activator (FSK) in Pkp3 KD SCC9 cells. Scale bar, 20 μm. (B) Immunofluorescence staining of 3D raft cultures after 6 d of differentiation shows recovery in DP distribution in Pkp3 KD rafts upon FSK treatment. Scale bar, 50 μm. (C) Immunofluorescence analysis of DP distribution in control and Pkp3-deficient SCC9 cells treated with vehicle, adrenergic agonist (ISO), or adrenergic antagonist (PROP). Scale bar, 20 μm. Note the recovery in ISO-treated Pkp3 KD and disruption of DP in PROP-treated control cells. (D) Cartoon depicting cAMP signaling with the activators and inhibitors used in this work. (E) Average fluorescence pixel intensities of DP at cell–cell borders of control and Pkp3 KD cells treated as specified in A (top, FSK) and C (bottom, ISO, PROP). Bars represent mean ± SEM. ns, p > 0.05, **0.001

    Techniques Used: Activation Assay, Immunofluorescence, Staining, Fluorescence

    Pkp3 is required for efficient assembly of DP into desmosomes. (A) Immunofluorescence staining for DP representing the distinct patterns of its disruption in Pkp2, Pkp3, and Pkp2-3 double-KD SCC9 cells. Scale bar, 20 μm. Yellow dashed rectangles in top images delineate areas enlarged at the bottom. Yellow arrows point to the sites of cell–cell contacts; red arrows point to DP-containing nonvesicular desmosome precursors in the cytoplasm. Note the absence of cytoplasmic particles in Pkp3 KD and both particles and cell–cell border DP in double-KD cells. (B) Average fluorescence DP pixel intensity at the cell–cell borders and inside the cytoplasm, measured for ≥100 individual cells, showed decreased DP at cell–cell borders in Pkp3 KD cells and a corresponding increase in the cytoplasm. Error bars represent ± SEM. *** p
    Figure Legend Snippet: Pkp3 is required for efficient assembly of DP into desmosomes. (A) Immunofluorescence staining for DP representing the distinct patterns of its disruption in Pkp2, Pkp3, and Pkp2-3 double-KD SCC9 cells. Scale bar, 20 μm. Yellow dashed rectangles in top images delineate areas enlarged at the bottom. Yellow arrows point to the sites of cell–cell contacts; red arrows point to DP-containing nonvesicular desmosome precursors in the cytoplasm. Note the absence of cytoplasmic particles in Pkp3 KD and both particles and cell–cell border DP in double-KD cells. (B) Average fluorescence DP pixel intensity at the cell–cell borders and inside the cytoplasm, measured for ≥100 individual cells, showed decreased DP at cell–cell borders in Pkp3 KD cells and a corresponding increase in the cytoplasm. Error bars represent ± SEM. *** p

    Techniques Used: Immunofluorescence, Staining, Fluorescence

    Model for the role of Pkp3 in cell–cell junction formation. Left, activation of cAMP pathway upon cell–cell contact in high calcium leads to rapid recruitment of Pkp3-Rap1 complex to E-cad. At the same time, juxtamembrane DP coalesces at the sites of cell–cell contacts, requiring either Pkp2 or Pkp3, forming nascent desmosomes. Functional Pkp3-Rap1-E-cad complex drives adherens junction maturation by pulling the cells closer together and desmosome assembly by signaling the formation of cytoplasmic DP particles. On the other hand, Pkp3 acts as a spacer, preventing the aberrant coalescence of nascent desmosomes at the membrane. Pkp2 harnesses the activity of PKCα and RhoA to facilitate actin-dependent transport of cytoplasmic DP particles to the membrane. Right, model of steady-state adherens and desmosome junctions demonstrating mature cortical actin distribution and correctly sealed adherens junctions in the presence of Pkp3. In the absence of Pkp3 (not shown), Pkp3-Rap1-E-cad complex fails to assemble, leading to immature adherens junctions and failure of DP cytoplasmic particles to form. In addition, aberrant coalescence of nascent desmosomes occurs at the membrane.
    Figure Legend Snippet: Model for the role of Pkp3 in cell–cell junction formation. Left, activation of cAMP pathway upon cell–cell contact in high calcium leads to rapid recruitment of Pkp3-Rap1 complex to E-cad. At the same time, juxtamembrane DP coalesces at the sites of cell–cell contacts, requiring either Pkp2 or Pkp3, forming nascent desmosomes. Functional Pkp3-Rap1-E-cad complex drives adherens junction maturation by pulling the cells closer together and desmosome assembly by signaling the formation of cytoplasmic DP particles. On the other hand, Pkp3 acts as a spacer, preventing the aberrant coalescence of nascent desmosomes at the membrane. Pkp2 harnesses the activity of PKCα and RhoA to facilitate actin-dependent transport of cytoplasmic DP particles to the membrane. Right, model of steady-state adherens and desmosome junctions demonstrating mature cortical actin distribution and correctly sealed adherens junctions in the presence of Pkp3. In the absence of Pkp3 (not shown), Pkp3-Rap1-E-cad complex fails to assemble, leading to immature adherens junctions and failure of DP cytoplasmic particles to form. In addition, aberrant coalescence of nascent desmosomes occurs at the membrane.

    Techniques Used: Activation Assay, Functional Assay, Activity Assay

    Pkp3-Rap1 complex is required for maintaining physical interaction between Rap1 and E-cad. (A) PLA (red dots) showing which of the molecular pairs indicated are in close proximity (within 100-nm range) within cells. Scale bar, 20 μm. (B) Western blot showing the immunoprecipitation (left) and total (right) protein levels of Pkp3, Rap1, E-cad, and Pkp2 for indicated conditions (with GAPDH as 5% loading control; asterisk indicates the Rap1 band just above the IgG light chain). Note the loss of E-cad from Rap1 complexes in Pkp3 KD condition. (C) Immunofluorescence staining showing the cell–cell border localization of E-cad in relation to DP (top) and Pkp3 (bottom). Note the minimal overlap between DP and E-cad and a substantial overlap between E-cad and Pkp3. Scale bar, 20 μm. (D) Western blot showing the immunoprecipitation (left) and total (right) protein levels of E-cad, Pkp3, Rap1, and EPAC for indicated conditions. Note that E-cad loss does not interfere with the interaction between Pkp3, Rap1, and EPAC. (E) Western blot showing the immunoprecipitation (left) and total (right) protein levels of E-cad, Pkp3, and Rap1 for indicated conditions. Note that the EPAC inhibitor ESI-09 causes the loss of E-cad but not Pkp3 from the Rap1 complex.
    Figure Legend Snippet: Pkp3-Rap1 complex is required for maintaining physical interaction between Rap1 and E-cad. (A) PLA (red dots) showing which of the molecular pairs indicated are in close proximity (within 100-nm range) within cells. Scale bar, 20 μm. (B) Western blot showing the immunoprecipitation (left) and total (right) protein levels of Pkp3, Rap1, E-cad, and Pkp2 for indicated conditions (with GAPDH as 5% loading control; asterisk indicates the Rap1 band just above the IgG light chain). Note the loss of E-cad from Rap1 complexes in Pkp3 KD condition. (C) Immunofluorescence staining showing the cell–cell border localization of E-cad in relation to DP (top) and Pkp3 (bottom). Note the minimal overlap between DP and E-cad and a substantial overlap between E-cad and Pkp3. Scale bar, 20 μm. (D) Western blot showing the immunoprecipitation (left) and total (right) protein levels of E-cad, Pkp3, Rap1, and EPAC for indicated conditions. Note that E-cad loss does not interfere with the interaction between Pkp3, Rap1, and EPAC. (E) Western blot showing the immunoprecipitation (left) and total (right) protein levels of E-cad, Pkp3, and Rap1 for indicated conditions. Note that the EPAC inhibitor ESI-09 causes the loss of E-cad but not Pkp3 from the Rap1 complex.

    Techniques Used: Proximity Ligation Assay, Western Blot, Immunoprecipitation, Immunofluorescence, Staining

    14) Product Images from "The mitochondrial outer-membrane location of the EXD2 exonuclease contradicts its direct role in nuclear DNA repair"

    Article Title: The mitochondrial outer-membrane location of the EXD2 exonuclease contradicts its direct role in nuclear DNA repair

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-23690-y

    EXD2 is accessible to added antibody in immunofluorescence without mitochondrial lysis. Immunofluorescent detection following paraformaldehyde fixation requires mitochondrial lysis using for example Triton X100. In the absence of this lysis step mitochondrial matrix proteins and for example mtDNA are not detectable. Thus (panel a) results shows that in the absence of TX100 lysis, EXD2 is detected while neither MRPL12 nor mtDNA are detected by IF. With TX100 lysis, all three are detected. A similar experiment (panel b) shows that both Tomm20 and EXD2, but not mtDNA are detected in the absence of TX100 lysis. A high resolution 20 × 30 µM subsection of a cell using the EXD2 and MRPL12 antibodies illustrates that the EXD2 signal is often enveloping the MRPL12 signal (panel c: some examples are indicated by a white arrow in the merged image), further illustrating EXD2 its outer-membrane localization.
    Figure Legend Snippet: EXD2 is accessible to added antibody in immunofluorescence without mitochondrial lysis. Immunofluorescent detection following paraformaldehyde fixation requires mitochondrial lysis using for example Triton X100. In the absence of this lysis step mitochondrial matrix proteins and for example mtDNA are not detectable. Thus (panel a) results shows that in the absence of TX100 lysis, EXD2 is detected while neither MRPL12 nor mtDNA are detected by IF. With TX100 lysis, all three are detected. A similar experiment (panel b) shows that both Tomm20 and EXD2, but not mtDNA are detected in the absence of TX100 lysis. A high resolution 20 × 30 µM subsection of a cell using the EXD2 and MRPL12 antibodies illustrates that the EXD2 signal is often enveloping the MRPL12 signal (panel c: some examples are indicated by a white arrow in the merged image), further illustrating EXD2 its outer-membrane localization.

    Techniques Used: Immunofluorescence, Lysis

    SiRNA mediated knockdown of EXD2 in U2OS cells results in a more widely spread mitochondrial network. In order to understand possible effects of EXD2 knockdown on mitochondrial network behaviour we labelled cells with EdU to identify cells in S-phase, in order to get a handle on mosaicism resulting from cell-cycle mediated effects on mitochondrial network dynamics (see main text). For this, cells were incubated for 30 min with EdU following transfection and 48–60 hrs incubation with either a pool of non-targeting control siRNAs (Csi) or a pool of three EXD2 stealth siRNAs (EXD2si3x). Following labelling for EdU incorporation (green, gr), cells were further labelled with antibodies for Tomm20 (red, r) and EXD2 (white). Control siRNA treated cells show frequent and strong perinuclear clustering of the mitochondrial network in particular in EdU positive cells. In contrast, the EXD2si3x typically show a more distributed and sometimes more hyperfused network. Two fields of view are shown for each condition.
    Figure Legend Snippet: SiRNA mediated knockdown of EXD2 in U2OS cells results in a more widely spread mitochondrial network. In order to understand possible effects of EXD2 knockdown on mitochondrial network behaviour we labelled cells with EdU to identify cells in S-phase, in order to get a handle on mosaicism resulting from cell-cycle mediated effects on mitochondrial network dynamics (see main text). For this, cells were incubated for 30 min with EdU following transfection and 48–60 hrs incubation with either a pool of non-targeting control siRNAs (Csi) or a pool of three EXD2 stealth siRNAs (EXD2si3x). Following labelling for EdU incorporation (green, gr), cells were further labelled with antibodies for Tomm20 (red, r) and EXD2 (white). Control siRNA treated cells show frequent and strong perinuclear clustering of the mitochondrial network in particular in EdU positive cells. In contrast, the EXD2si3x typically show a more distributed and sometimes more hyperfused network. Two fields of view are shown for each condition.

    Techniques Used: Incubation, Transfection

    EXD2 is a mitochondrial outer-membrane associated protein. U2OS cell crude mitochondria isolated by differential centrifugation and pure nuclei isolated on iodixanol gradients were tested for EXD2 abundance in control siRNA and EXD2 siRNA treated cells (panel a1). Results show that the vast majority of EXD2, similar to mitochondrial marker proteins mtSSB and porin are found in the mitochondrial fraction and not in the nuclear fraction, in which the nuclear marker nucleophosmin is identified. SiRNA treatment confirms the identity of the full length (fl) ~70 kDa EXD2 protein and several lower abundant species of 30 kDa and higher molecular weight. Knockdown with either the combined pool of three commercial siRNAs or each individual siRNA show similar knockdown efficiencies in total cell lysates of U2OS cells on Western blots (panel a2). At the same time, several lower molecular weight EXD2 species are also identified and all appear to be equally sensitive to each individual siRNA suggesting they might be breakdown or processed EXD2 forms. Protease protection demonstrates mitochondrial EXD2 is mostly found in the mitochondrial outer-membrane (panel b). Mitochondria and digitonin-derived mitoplasts from HEK293 cells were treated either with Proteinase K (ProtK) alone or with ProtK and Triton-X100 (TX100) to lyse the inner and outer membrane. Results show that whereas TFAM, an mtDNA associated protein, is protected from ProtK in the absence of TX100, EXD2 is not, suggesting and outer-membrane localization. *Indicates a remnant signal from the probing with an antibody against a different mitochondrial candidate protein not relevant for this paper. A Na 2 CO 3 extract of crude mitochondria from HEK293 cells (panel c) shows that similar to cytochrome c oxidase subunit I (an integral membrane protein), full length EXD2 is found predominantly in the pellet (membrane) fraction, whereas the majority of HSP60 is found in the supernatant (non-membrane) fraction. For each panel (except panel b) cropped images show the results of incubations with subsequent antibodies on the same blots, indicated by dividing lines (see Supplementary info for full blot images).
    Figure Legend Snippet: EXD2 is a mitochondrial outer-membrane associated protein. U2OS cell crude mitochondria isolated by differential centrifugation and pure nuclei isolated on iodixanol gradients were tested for EXD2 abundance in control siRNA and EXD2 siRNA treated cells (panel a1). Results show that the vast majority of EXD2, similar to mitochondrial marker proteins mtSSB and porin are found in the mitochondrial fraction and not in the nuclear fraction, in which the nuclear marker nucleophosmin is identified. SiRNA treatment confirms the identity of the full length (fl) ~70 kDa EXD2 protein and several lower abundant species of 30 kDa and higher molecular weight. Knockdown with either the combined pool of three commercial siRNAs or each individual siRNA show similar knockdown efficiencies in total cell lysates of U2OS cells on Western blots (panel a2). At the same time, several lower molecular weight EXD2 species are also identified and all appear to be equally sensitive to each individual siRNA suggesting they might be breakdown or processed EXD2 forms. Protease protection demonstrates mitochondrial EXD2 is mostly found in the mitochondrial outer-membrane (panel b). Mitochondria and digitonin-derived mitoplasts from HEK293 cells were treated either with Proteinase K (ProtK) alone or with ProtK and Triton-X100 (TX100) to lyse the inner and outer membrane. Results show that whereas TFAM, an mtDNA associated protein, is protected from ProtK in the absence of TX100, EXD2 is not, suggesting and outer-membrane localization. *Indicates a remnant signal from the probing with an antibody against a different mitochondrial candidate protein not relevant for this paper. A Na 2 CO 3 extract of crude mitochondria from HEK293 cells (panel c) shows that similar to cytochrome c oxidase subunit I (an integral membrane protein), full length EXD2 is found predominantly in the pellet (membrane) fraction, whereas the majority of HSP60 is found in the supernatant (non-membrane) fraction. For each panel (except panel b) cropped images show the results of incubations with subsequent antibodies on the same blots, indicated by dividing lines (see Supplementary info for full blot images).

    Techniques Used: Isolation, Centrifugation, Marker, Molecular Weight, Western Blot, Derivative Assay

    Nuclear DNA-damage does not relocate EXD2 to the nucleus. (panel a) HeLa cells were treated with 13 Gy X-irradiation to induce nuclear DNA damage and allowed to recover for 30 minutes. Results show that H2AX phosphorylation is considerably increased in the nucleus following this damage whereas EXD2 retains its predominant mitochondrial localization similar to its location in control cells. (panel b) U2OS cells were treated with 250 µg/ml Zeocin for 1 hr and allowed to recover for 1 hr after replacing the Zeocin-containing medium with regular medium. Cells were then processed for IF detection of γH2AX and EXD2 and show a similar result as the X-irradiation in HeLa cells.
    Figure Legend Snippet: Nuclear DNA-damage does not relocate EXD2 to the nucleus. (panel a) HeLa cells were treated with 13 Gy X-irradiation to induce nuclear DNA damage and allowed to recover for 30 minutes. Results show that H2AX phosphorylation is considerably increased in the nucleus following this damage whereas EXD2 retains its predominant mitochondrial localization similar to its location in control cells. (panel b) U2OS cells were treated with 250 µg/ml Zeocin for 1 hr and allowed to recover for 1 hr after replacing the Zeocin-containing medium with regular medium. Cells were then processed for IF detection of γH2AX and EXD2 and show a similar result as the X-irradiation in HeLa cells.

    Techniques Used: Irradiation

    Knockdown or overexpression in U2OS cells of full-length EXD2 confirms the mitochondrial localization of EXD2. ProteinAtlas describes their EXD2 antibody, which we have used throughout this study, as having a mitochondrial and possible intermediate filament localization. To test the localization and the validity of their antibody we tested the EXD2 antibody, together with an antibody against the outer-membrane protein Tomm20 and an antibody against the intermediate filament protein vimentin (Vim) using immunofluorescence following transfection with either a pool of non-targeting control siRNAs or a pool of three EXD2 Stealth siRNAs (panel a). Co-staining in control siRNA cells with Tomm20 and vimentin shows co-localization of the EXD2 signal both with mitochondrial and intermediate filament signals. EXD2 siRNA treatment shows that while the EXD2 mitochondrial signal is no longer observed, the intermediate filament signal remains suggesting that this signal is either non-specific or that the siRNA pool used does not affect intermediate filament associated EXD2. Transient overexpression of the predicted full length protein, either w/o a tag or with a C-terminal combined Myc/FLAG tag shows an exclusive mitochondrial localization of the protein as illustrated by Tomm20 co-staining, while higher level overexpression results in mitochondrial perinuclear clustering (panel b). With very high overexpression, the whole mitochondrial network collapsed in one large perinuclear cluster that had lost any typical mitochondrial network-like structure (Supplementary Fig. S1 ). Overexpressed full length EXD2 showed no evidence of either nuclear or intermediate filament localization. Please note that for this Figure images have been selected deliberately to best illustrate the mitochondrial localization of EXD2. Image views have thus been chosen showing cells with a clear and extended mitochondrial network, while trying to avoid cells with a condensed/collapsed mitochondrial network.
    Figure Legend Snippet: Knockdown or overexpression in U2OS cells of full-length EXD2 confirms the mitochondrial localization of EXD2. ProteinAtlas describes their EXD2 antibody, which we have used throughout this study, as having a mitochondrial and possible intermediate filament localization. To test the localization and the validity of their antibody we tested the EXD2 antibody, together with an antibody against the outer-membrane protein Tomm20 and an antibody against the intermediate filament protein vimentin (Vim) using immunofluorescence following transfection with either a pool of non-targeting control siRNAs or a pool of three EXD2 Stealth siRNAs (panel a). Co-staining in control siRNA cells with Tomm20 and vimentin shows co-localization of the EXD2 signal both with mitochondrial and intermediate filament signals. EXD2 siRNA treatment shows that while the EXD2 mitochondrial signal is no longer observed, the intermediate filament signal remains suggesting that this signal is either non-specific or that the siRNA pool used does not affect intermediate filament associated EXD2. Transient overexpression of the predicted full length protein, either w/o a tag or with a C-terminal combined Myc/FLAG tag shows an exclusive mitochondrial localization of the protein as illustrated by Tomm20 co-staining, while higher level overexpression results in mitochondrial perinuclear clustering (panel b). With very high overexpression, the whole mitochondrial network collapsed in one large perinuclear cluster that had lost any typical mitochondrial network-like structure (Supplementary Fig. S1 ). Overexpressed full length EXD2 showed no evidence of either nuclear or intermediate filament localization. Please note that for this Figure images have been selected deliberately to best illustrate the mitochondrial localization of EXD2. Image views have thus been chosen showing cells with a clear and extended mitochondrial network, while trying to avoid cells with a condensed/collapsed mitochondrial network.

    Techniques Used: Over Expression, Immunofluorescence, Transfection, Staining, FLAG-tag

    15) Product Images from "Human Negative Elongation Factor Activates Transcription and Regulates Alternative Transcription Initiation *"

    Article Title: Human Negative Elongation Factor Activates Transcription and Regulates Alternative Transcription Initiation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.084285

    Exon array-based profiling of gene expression in NELF knockdown cells. A , Western blot analysis of NELF expression in T47D cells transfected with siRNA oligonucleotides for control luciferase (siControl) or the individual NELF subunits. α-Tubulin
    Figure Legend Snippet: Exon array-based profiling of gene expression in NELF knockdown cells. A , Western blot analysis of NELF expression in T47D cells transfected with siRNA oligonucleotides for control luciferase (siControl) or the individual NELF subunits. α-Tubulin

    Techniques Used: Expressing, Western Blot, Transfection, Luciferase

    NELF depletion increases total histone density and decreases activation-associated histone modifications at NELF target genes. T47D cells transfected with control and NELF-E siRNA oligonucleotides were used for ChIP with antibodies recognizing total histone
    Figure Legend Snippet: NELF depletion increases total histone density and decreases activation-associated histone modifications at NELF target genes. T47D cells transfected with control and NELF-E siRNA oligonucleotides were used for ChIP with antibodies recognizing total histone

    Techniques Used: Activation Assay, Transfection, Chromatin Immunoprecipitation

    NELF is directly involved in transcription of a number of cell cycle-associated genes. A , real time PCR analysis of gene expression in control and NELF knockdown cells. T47D cells were transfected with siRNA oligonucleotides for 4 days. Total RNA was
    Figure Legend Snippet: NELF is directly involved in transcription of a number of cell cycle-associated genes. A , real time PCR analysis of gene expression in control and NELF knockdown cells. T47D cells were transfected with siRNA oligonucleotides for 4 days. Total RNA was

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

    NELF depletion delays cell cycle progression. A , effect of NELF knockdown on cell proliferation. T47D cells were transfected with siRNA oligonucleotides against the individual NELF subunits, and the cell number was counted on days 2, 3, and 4 after siRNA
    Figure Legend Snippet: NELF depletion delays cell cycle progression. A , effect of NELF knockdown on cell proliferation. T47D cells were transfected with siRNA oligonucleotides against the individual NELF subunits, and the cell number was counted on days 2, 3, and 4 after siRNA

    Techniques Used: Transfection

    16) Product Images from "P0-Related Protein Accelerates Human Mesenchymal Stromal Cell Migration by Modulating VLA-5 Interactions with Fibronectin"

    Article Title: P0-Related Protein Accelerates Human Mesenchymal Stromal Cell Migration by Modulating VLA-5 Interactions with Fibronectin

    Journal: Cells

    doi: 10.3390/cells9051100

    Human PZR regulates the migration of murine NIH3T3-PZR transfectants on fibronectin. ( A ) Flow cytometric histograms showing the level of expression of human PZR and PZRb as analyzed using WM78 plus FITC-goat anti-mIgG1 staining of NIH3T3 ( a ), NIH3T3-PZR ( b ), and NIH3T3-PZRb ( c ) (black outlined histograms). Grey outlined histograms are isotype matched mIgG1 negative control staining. ( B ) Graphical representation of WM78 staining in ( A ) showing MFI values as means ± S.E.M. for triplicate stains. ( C ) Representative WM78 followed by FITC-goat anti-mIgG1 staining (green) of NIH3T3-PZR stable transfectants ( a ), NIH3T3–PZRb stable transfectants ( b ), and mock transfected NIH3T3 cells ( c ) and analysis using confocal microscopy. Panels ( d ) and ( e ) show a lack of surface staining of NIH3T3-PZR and NIH3T3-PZRb cells with FITC-goat anti-mIgG1 FITC as negative controls. ( D ) Western blots of human PZR isoforms from NIH3T3 transfectants. Biotinylated cell lysates were immunoprecipitated with WM78, separated by 12% polyacrylamide gel electrophoresis and proteins detected with streptavidin alkaline phosphatase (SA-HRP). ( E ) NIH3T3 and NIH3T3 stably expressing human PZR or PZRb were stained with the biotin-conjugated rat anti-mouse -CD29, -CD49e, -CD49d, -CD51, or rat isotype controls, followed by Alexa 546-streptavidin and analyzed by flow cytometry. For ( B ) and ( E ), values represent means ± S.E.M. MFIs for three independent experiments. ( F ) Percentage migration (at 6 h) of input NIH3T3-PZR, -PZRb, and NIH3T3 non-transduced cells on fibronectin, collagen IV, or laminin or in the absence of ECM. PZR enhanced migration on FN compared to non-ECM control ( p
    Figure Legend Snippet: Human PZR regulates the migration of murine NIH3T3-PZR transfectants on fibronectin. ( A ) Flow cytometric histograms showing the level of expression of human PZR and PZRb as analyzed using WM78 plus FITC-goat anti-mIgG1 staining of NIH3T3 ( a ), NIH3T3-PZR ( b ), and NIH3T3-PZRb ( c ) (black outlined histograms). Grey outlined histograms are isotype matched mIgG1 negative control staining. ( B ) Graphical representation of WM78 staining in ( A ) showing MFI values as means ± S.E.M. for triplicate stains. ( C ) Representative WM78 followed by FITC-goat anti-mIgG1 staining (green) of NIH3T3-PZR stable transfectants ( a ), NIH3T3–PZRb stable transfectants ( b ), and mock transfected NIH3T3 cells ( c ) and analysis using confocal microscopy. Panels ( d ) and ( e ) show a lack of surface staining of NIH3T3-PZR and NIH3T3-PZRb cells with FITC-goat anti-mIgG1 FITC as negative controls. ( D ) Western blots of human PZR isoforms from NIH3T3 transfectants. Biotinylated cell lysates were immunoprecipitated with WM78, separated by 12% polyacrylamide gel electrophoresis and proteins detected with streptavidin alkaline phosphatase (SA-HRP). ( E ) NIH3T3 and NIH3T3 stably expressing human PZR or PZRb were stained with the biotin-conjugated rat anti-mouse -CD29, -CD49e, -CD49d, -CD51, or rat isotype controls, followed by Alexa 546-streptavidin and analyzed by flow cytometry. For ( B ) and ( E ), values represent means ± S.E.M. MFIs for three independent experiments. ( F ) Percentage migration (at 6 h) of input NIH3T3-PZR, -PZRb, and NIH3T3 non-transduced cells on fibronectin, collagen IV, or laminin or in the absence of ECM. PZR enhanced migration on FN compared to non-ECM control ( p

    Techniques Used: Migration, Expressing, Staining, Negative Control, Transfection, Confocal Microscopy, Western Blot, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Stable Transfection, Flow Cytometry

    Co-localization of PZR and integrins on migrating NIH3T3 transfectants and hBM MSCs. ( Ai ) Representative FACS histograms of hBM MSCs stained for CD29 (β1 integrin), CD49e (α5 integrin), CD49d (α4 integrin), CD51 (vitronectin receptor), or the relevant isotype control (mIgG1) followed by Alexa-488 goat anti-mIgG1. MFI ± S.E.M. shown above histograms ( n = 3 independent experiments). Black histograms: integrin staining; white histograms: mIgG1 negative control. ( Aii ) hBM MSCs were untreated or incubated with blocking antibodies against CD29, CD49e, CD49d, CD51, and CD51/61 or the corresponding isotype controls before being allowed to adhere to fibronectin (FN) or the negative control, BSA. Values are means ± S.E.M. for three independent experiments performed in triplicate. ( B ) Migration assay using NIH3T3-hPZR ( a ) with one area analyzed by confocal microscopy circled in red. ( b,c ) NIH3T3-PZR transfectants from the 6 h migratory interface double stained with WM78 (PZR; green stain) and biotin-CD29 (red stain) or ( d ) biotin-CD49e (red stain) plus appropriate secondary fluorescent reagents. hBM MSCs from the 6 h migratory interface double stained with WM78 and Alexa488 goat anti mIgG1 (PZR; green stain), then blocked with mIgG1 and stained with biotinylated ( e ) CD29 (red stain) or ( f ) CD49e (red stain) with streptavidin-conjugated Alexa 546. There is a co-association of PZR with α5 (CD49e) or β1 (CD29) at the leading edge of the migrating cell. C ( a ) Quantitation of PZR co-localizing with CD29 and CD49e or CD49d and CD51 in 6 h migrating hBM MSCs. Values are means ± S.E.M. for three independent experiments performed in triplicate. Inset shows co-immuno-precipitation and Western blotting with respective the anti-PZR WM78 mAb and biotin conjugated anti-human CD49e or CD29, and using mIgG1 as the negative control for the immunoprecipitation (i.p.). Lane A: hBM MSC cell lysate, lane B: hBM MSC i.p with anti-PZR, lane C: hBM MSC i.p. with negative control. C ( b ) Confocal microscopy of PZR co-localizing with CD29 and CD49e and to a much lesser extent with CD49d and CD51 in NIH3T3-PZR after 6 h of migration. Inset shows co-immunoprecipitation and Western blotting of PZR (WM78 mAb) with biotin anti-mouse CD49e or CD29. mIgG1 was used as negative i.p. control. Lane A: NIH3T3-PZR cell lysate, lane B: NIH3T3-PZR i.p with WM78, lane C: NIH3T3-PZRb i.p. with WM78, lane D: NIH3T3 i.p. with WM78 and lane E: NIH3T3-hPZR i.p. with mIgG1.
    Figure Legend Snippet: Co-localization of PZR and integrins on migrating NIH3T3 transfectants and hBM MSCs. ( Ai ) Representative FACS histograms of hBM MSCs stained for CD29 (β1 integrin), CD49e (α5 integrin), CD49d (α4 integrin), CD51 (vitronectin receptor), or the relevant isotype control (mIgG1) followed by Alexa-488 goat anti-mIgG1. MFI ± S.E.M. shown above histograms ( n = 3 independent experiments). Black histograms: integrin staining; white histograms: mIgG1 negative control. ( Aii ) hBM MSCs were untreated or incubated with blocking antibodies against CD29, CD49e, CD49d, CD51, and CD51/61 or the corresponding isotype controls before being allowed to adhere to fibronectin (FN) or the negative control, BSA. Values are means ± S.E.M. for three independent experiments performed in triplicate. ( B ) Migration assay using NIH3T3-hPZR ( a ) with one area analyzed by confocal microscopy circled in red. ( b,c ) NIH3T3-PZR transfectants from the 6 h migratory interface double stained with WM78 (PZR; green stain) and biotin-CD29 (red stain) or ( d ) biotin-CD49e (red stain) plus appropriate secondary fluorescent reagents. hBM MSCs from the 6 h migratory interface double stained with WM78 and Alexa488 goat anti mIgG1 (PZR; green stain), then blocked with mIgG1 and stained with biotinylated ( e ) CD29 (red stain) or ( f ) CD49e (red stain) with streptavidin-conjugated Alexa 546. There is a co-association of PZR with α5 (CD49e) or β1 (CD29) at the leading edge of the migrating cell. C ( a ) Quantitation of PZR co-localizing with CD29 and CD49e or CD49d and CD51 in 6 h migrating hBM MSCs. Values are means ± S.E.M. for three independent experiments performed in triplicate. Inset shows co-immuno-precipitation and Western blotting with respective the anti-PZR WM78 mAb and biotin conjugated anti-human CD49e or CD29, and using mIgG1 as the negative control for the immunoprecipitation (i.p.). Lane A: hBM MSC cell lysate, lane B: hBM MSC i.p with anti-PZR, lane C: hBM MSC i.p. with negative control. C ( b ) Confocal microscopy of PZR co-localizing with CD29 and CD49e and to a much lesser extent with CD49d and CD51 in NIH3T3-PZR after 6 h of migration. Inset shows co-immunoprecipitation and Western blotting of PZR (WM78 mAb) with biotin anti-mouse CD49e or CD29. mIgG1 was used as negative i.p. control. Lane A: NIH3T3-PZR cell lysate, lane B: NIH3T3-PZR i.p with WM78, lane C: NIH3T3-PZRb i.p. with WM78, lane D: NIH3T3 i.p. with WM78 and lane E: NIH3T3-hPZR i.p. with mIgG1.

    Techniques Used: FACS, Staining, Negative Control, Incubation, Blocking Assay, Migration, Confocal Microscopy, Quantitation Assay, Immunoprecipitation, Western Blot

    17) Product Images from "Regulation of α2B-Adrenergic Receptor Cell Surface Transport by GGA1 and GGA2"

    Article Title: Regulation of α2B-Adrenergic Receptor Cell Surface Transport by GGA1 and GGA2

    Journal: Scientific Reports

    doi: 10.1038/srep37921

    Effect of GGA1 and GGA2 depletion on the dendritic expression of α 2B -AR in primary cortical neurons. ( A ) Effect of GGA1 knockdown on α 2B -AR expression in the dendrites of primary cortical neurons. The cortical neurons were transfected with α 2B -AR-GFP together with GGA1 siRNA at DIV 5. Two days after transfection, the neurons were stained with antibodies against GGA1. The distribution of α 2B -AR was visualized by confocal microscopy. ( B ) Effect of GGA2 knockdown on the dendritic expression of α 2B -AR. The data shown are representative images in at least 4 individual experiments. Arrows indicate the expression of GGA1 or GGA2. Scale bars, 20 μm. ( C ) Quantitative data shown in ( A , B ) (n = 17). α 2B -AR expression in the dendrites was determined by measuring the GFP signal. * p
    Figure Legend Snippet: Effect of GGA1 and GGA2 depletion on the dendritic expression of α 2B -AR in primary cortical neurons. ( A ) Effect of GGA1 knockdown on α 2B -AR expression in the dendrites of primary cortical neurons. The cortical neurons were transfected with α 2B -AR-GFP together with GGA1 siRNA at DIV 5. Two days after transfection, the neurons were stained with antibodies against GGA1. The distribution of α 2B -AR was visualized by confocal microscopy. ( B ) Effect of GGA2 knockdown on the dendritic expression of α 2B -AR. The data shown are representative images in at least 4 individual experiments. Arrows indicate the expression of GGA1 or GGA2. Scale bars, 20 μm. ( C ) Quantitative data shown in ( A , B ) (n = 17). α 2B -AR expression in the dendrites was determined by measuring the GFP signal. * p

    Techniques Used: Expressing, Transfection, Staining, Confocal Microscopy

    Inhibition of cell surface expression of α 2B -AR by siRNA-mediated depletion of GGA1 and GGA2. ( A ) siRNA-mediated depletion of GGA1 and GGA2 in HEK293 cells. ( B ) Effect of siRNA-mediated knockdown of GGA1 and GGA2 on the cell surface expression of α 2B -AR. HEK293 cells inducibly expressing α 2B -AR were transfected with control siRNA or siRNA targeting GGA1 and GGA2 and incubated with doxycycline as described in legends of Fig. 1B . The average specific binding of [ 3 H]-RX821002 from cells without siRNA transfection and treated with doxycycline for 28 h was 34,423 ± 563 cpm per well. ( C ) Effect of combination knockdown of GGA1, GGA2 and GGA3 on the cell surface expression of α 2B -AR in HEK293 cells. ( D ) Effect of knockdown of GGA1, GGA2 and GGA3 on the Golgi structure. HEK293 cells were transfected with control or GGA siRNA for 48 h and then stained with antibodies against GM130 (1:200 dilution) and p230 (1:100 dilution) overnight. Scale bar, 10 μm. ( E ) Effect of GGA1 and GGA2 knockdown on total α 2B -AR expression. HEK293 cells inducibly expressing α 2B -AR were transfected with control or GGA shRNA or siRNA for 24 h and incubated with doxycycline (40 ng/ml) for another 24 h. The overall α 2B -AR expression was measured by flow cytometry following staining with HA antibodies in permeabilized cells (n = 3). ( F ) Effect of GGA1 and GGA2 knockdown on the internalization of α 2B -AR. HEK293 cells stably expressing α 2B -AR were transfected with arrestin-3 and control or GGA shRNA and incubated with doxycycline as described above. The cells were then stimulated with epinephrine (100 μM) for 10, 20 and 30 min (n = 3). The cell surface expression of α 2B -AR was determined by intact cell ligand binding using [ 3 H]-RX821002. The data are presented as the mean ± S.E. of at least three individual experiments in ( B , C , E , F ). * p
    Figure Legend Snippet: Inhibition of cell surface expression of α 2B -AR by siRNA-mediated depletion of GGA1 and GGA2. ( A ) siRNA-mediated depletion of GGA1 and GGA2 in HEK293 cells. ( B ) Effect of siRNA-mediated knockdown of GGA1 and GGA2 on the cell surface expression of α 2B -AR. HEK293 cells inducibly expressing α 2B -AR were transfected with control siRNA or siRNA targeting GGA1 and GGA2 and incubated with doxycycline as described in legends of Fig. 1B . The average specific binding of [ 3 H]-RX821002 from cells without siRNA transfection and treated with doxycycline for 28 h was 34,423 ± 563 cpm per well. ( C ) Effect of combination knockdown of GGA1, GGA2 and GGA3 on the cell surface expression of α 2B -AR in HEK293 cells. ( D ) Effect of knockdown of GGA1, GGA2 and GGA3 on the Golgi structure. HEK293 cells were transfected with control or GGA siRNA for 48 h and then stained with antibodies against GM130 (1:200 dilution) and p230 (1:100 dilution) overnight. Scale bar, 10 μm. ( E ) Effect of GGA1 and GGA2 knockdown on total α 2B -AR expression. HEK293 cells inducibly expressing α 2B -AR were transfected with control or GGA shRNA or siRNA for 24 h and incubated with doxycycline (40 ng/ml) for another 24 h. The overall α 2B -AR expression was measured by flow cytometry following staining with HA antibodies in permeabilized cells (n = 3). ( F ) Effect of GGA1 and GGA2 knockdown on the internalization of α 2B -AR. HEK293 cells stably expressing α 2B -AR were transfected with arrestin-3 and control or GGA shRNA and incubated with doxycycline as described above. The cells were then stimulated with epinephrine (100 μM) for 10, 20 and 30 min (n = 3). The cell surface expression of α 2B -AR was determined by intact cell ligand binding using [ 3 H]-RX821002. The data are presented as the mean ± S.E. of at least three individual experiments in ( B , C , E , F ). * p

    Techniques Used: Inhibition, Expressing, Transfection, Incubation, Binding Assay, Staining, shRNA, Flow Cytometry, Cytometry, Stable Transfection, Ligand Binding Assay

    Inhibition of cell surface expression of α 2B -AR by shRNA-mediated knockdown of GGA1 and GGA2. ( A ) shRNA-mediated depletion of GGA1 and GGA2 in HEK293 cells. The expression of GGAs was measured by immunoblotting using isoform-specific antibodies. ( B ) Effect of shRNA-mediated knockdown of GGA1 and GGA2 on the cell surface expression of α 2B -AR. HEK293 cells inducibly expressing α 2B -AR were transfected with control or GGA shRNA and then incubated with doxycycline at the concentration of 40 ng/ml for different time periods (0, 4, 8, 12, 16, 20, 24 and 28 h). The cell surface expression of α 2B -AR was determined by intact cell ligand binding using [ 3 H]-RX821002. The data shown are percentages of specific binding obtained from cells transfected with control shRNA and treated with doxycycline for 28 h, in which the mean value of specific [ 3 H]-RX821002 binding was 35,642 ± 985 cpm per well (n = 4) and presented as the mean ± S.E. of at least three individual experiments. * p
    Figure Legend Snippet: Inhibition of cell surface expression of α 2B -AR by shRNA-mediated knockdown of GGA1 and GGA2. ( A ) shRNA-mediated depletion of GGA1 and GGA2 in HEK293 cells. The expression of GGAs was measured by immunoblotting using isoform-specific antibodies. ( B ) Effect of shRNA-mediated knockdown of GGA1 and GGA2 on the cell surface expression of α 2B -AR. HEK293 cells inducibly expressing α 2B -AR were transfected with control or GGA shRNA and then incubated with doxycycline at the concentration of 40 ng/ml for different time periods (0, 4, 8, 12, 16, 20, 24 and 28 h). The cell surface expression of α 2B -AR was determined by intact cell ligand binding using [ 3 H]-RX821002. The data shown are percentages of specific binding obtained from cells transfected with control shRNA and treated with doxycycline for 28 h, in which the mean value of specific [ 3 H]-RX821002 binding was 35,642 ± 985 cpm per well (n = 4) and presented as the mean ± S.E. of at least three individual experiments. * p

    Techniques Used: Inhibition, Expressing, shRNA, Transfection, Incubation, Concentration Assay, Ligand Binding Assay, Binding Assay

    18) Product Images from "Regulation of α2B-Adrenergic Receptor Cell Surface Transport by GGA1 and GGA2"

    Article Title: Regulation of α2B-Adrenergic Receptor Cell Surface Transport by GGA1 and GGA2

    Journal: Scientific Reports

    doi: 10.1038/srep37921

    Inhibition of cell surface expression of α 2B -AR by siRNA-mediated depletion of GGA1 and GGA2. ( A ) siRNA-mediated depletion of GGA1 and GGA2 in HEK293 cells. ( B ) Effect of siRNA-mediated knockdown of GGA1 and GGA2 on the cell surface expression of α 2B -AR. HEK293 cells inducibly expressing α 2B -AR were transfected with control siRNA or siRNA targeting GGA1 and GGA2 and incubated with doxycycline as described in legends of Fig. 1B . The average specific binding of [ 3 H]-RX821002 from cells without siRNA transfection and treated with doxycycline for 28 h was 34,423 ± 563 cpm per well. ( C ) Effect of combination knockdown of GGA1, GGA2 and GGA3 on the cell surface expression of α 2B -AR in HEK293 cells. ( D ) Effect of knockdown of GGA1, GGA2 and GGA3 on the Golgi structure. HEK293 cells were transfected with control or GGA siRNA for 48 h and then stained with antibodies against GM130 (1:200 dilution) and p230 (1:100 dilution) overnight. Scale bar, 10 μm. ( E ) Effect of GGA1 and GGA2 knockdown on total α 2B -AR expression. HEK293 cells inducibly expressing α 2B -AR were transfected with control or GGA shRNA or siRNA for 24 h and incubated with doxycycline (40 ng/ml) for another 24 h. The overall α 2B -AR expression was measured by flow cytometry following staining with HA antibodies in permeabilized cells (n = 3). ( F ) Effect of GGA1 and GGA2 knockdown on the internalization of α 2B -AR. HEK293 cells stably expressing α 2B -AR were transfected with arrestin-3 and control or GGA shRNA and incubated with doxycycline as described above. The cells were then stimulated with epinephrine (100 μM) for 10, 20 and 30 min (n = 3). The cell surface expression of α 2B -AR was determined by intact cell ligand binding using [ 3 H]-RX821002. The data are presented as the mean ± S.E. of at least three individual experiments in ( B , C , E , F ). * p
    Figure Legend Snippet: Inhibition of cell surface expression of α 2B -AR by siRNA-mediated depletion of GGA1 and GGA2. ( A ) siRNA-mediated depletion of GGA1 and GGA2 in HEK293 cells. ( B ) Effect of siRNA-mediated knockdown of GGA1 and GGA2 on the cell surface expression of α 2B -AR. HEK293 cells inducibly expressing α 2B -AR were transfected with control siRNA or siRNA targeting GGA1 and GGA2 and incubated with doxycycline as described in legends of Fig. 1B . The average specific binding of [ 3 H]-RX821002 from cells without siRNA transfection and treated with doxycycline for 28 h was 34,423 ± 563 cpm per well. ( C ) Effect of combination knockdown of GGA1, GGA2 and GGA3 on the cell surface expression of α 2B -AR in HEK293 cells. ( D ) Effect of knockdown of GGA1, GGA2 and GGA3 on the Golgi structure. HEK293 cells were transfected with control or GGA siRNA for 48 h and then stained with antibodies against GM130 (1:200 dilution) and p230 (1:100 dilution) overnight. Scale bar, 10 μm. ( E ) Effect of GGA1 and GGA2 knockdown on total α 2B -AR expression. HEK293 cells inducibly expressing α 2B -AR were transfected with control or GGA shRNA or siRNA for 24 h and incubated with doxycycline (40 ng/ml) for another 24 h. The overall α 2B -AR expression was measured by flow cytometry following staining with HA antibodies in permeabilized cells (n = 3). ( F ) Effect of GGA1 and GGA2 knockdown on the internalization of α 2B -AR. HEK293 cells stably expressing α 2B -AR were transfected with arrestin-3 and control or GGA shRNA and incubated with doxycycline as described above. The cells were then stimulated with epinephrine (100 μM) for 10, 20 and 30 min (n = 3). The cell surface expression of α 2B -AR was determined by intact cell ligand binding using [ 3 H]-RX821002. The data are presented as the mean ± S.E. of at least three individual experiments in ( B , C , E , F ). * p

    Techniques Used: Inhibition, Expressing, Transfection, Incubation, Binding Assay, Staining, shRNA, Flow Cytometry, Cytometry, Stable Transfection, Ligand Binding Assay

    Identification of the GGA1- and GGA2-binding sites in the α 2B -AR ICL3 by progressive deletion. ( A ) Interaction of different ICL3 fragments with the GGA1 hinge and the GGA2 GAE domains. Each ICL3 fragment was generated as GST fusion proteins. The GGA1 hinge and the GGA2 GAE domains were generated as GFP fusion proteins. Their interactions were determined in GST fusion protein pulldown assays. Bound GGA domains were revealed by immunoblotting using GFP antibodies. Bottom panel shows Coomassie blue staining of purified GST fusion proteins. Similar results were obtained in at least three different experiments. The blots from two gels that were run under the same experimental conditions were combined to show the interaction of the GGA1 hinge with different ICL3 domains (upper panel). ( B ) A summary of progressive deletion to identify the GGA1- and GGA2-binding domains in the α 2B -AR ICL3 as shown in ( A ). +Interacting with individual GGA domains; −, not interacting with GGA. ( C ) A diagram showing differential interactions between α 2B -AR and three GGAs. The GGA1 hinge and the GGA2 GAE domains bind to two subdomains of the α 2B -AR ICL3 as revealed in the current studies, whereas the GGA3 VHS domain interacts with the α 2B -AR ICL3, specifically the 3R motif, as demonstrated in our previous studies 47 .
    Figure Legend Snippet: Identification of the GGA1- and GGA2-binding sites in the α 2B -AR ICL3 by progressive deletion. ( A ) Interaction of different ICL3 fragments with the GGA1 hinge and the GGA2 GAE domains. Each ICL3 fragment was generated as GST fusion proteins. The GGA1 hinge and the GGA2 GAE domains were generated as GFP fusion proteins. Their interactions were determined in GST fusion protein pulldown assays. Bound GGA domains were revealed by immunoblotting using GFP antibodies. Bottom panel shows Coomassie blue staining of purified GST fusion proteins. Similar results were obtained in at least three different experiments. The blots from two gels that were run under the same experimental conditions were combined to show the interaction of the GGA1 hinge with different ICL3 domains (upper panel). ( B ) A summary of progressive deletion to identify the GGA1- and GGA2-binding domains in the α 2B -AR ICL3 as shown in ( A ). +Interacting with individual GGA domains; −, not interacting with GGA. ( C ) A diagram showing differential interactions between α 2B -AR and three GGAs. The GGA1 hinge and the GGA2 GAE domains bind to two subdomains of the α 2B -AR ICL3 as revealed in the current studies, whereas the GGA3 VHS domain interacts with the α 2B -AR ICL3, specifically the 3R motif, as demonstrated in our previous studies 47 .

    Techniques Used: Binding Assay, Generated, Staining, Purification

    19) Product Images from "Regulation of α2B-Adrenergic Receptor Cell Surface Transport by GGA1 and GGA2"

    Article Title: Regulation of α2B-Adrenergic Receptor Cell Surface Transport by GGA1 and GGA2

    Journal: Scientific Reports

    doi: 10.1038/srep37921

    Interaction of α 2B -AR with GGA1 and GGA2. ( A ) Interaction of α 2B -AR with GGA1 and GGA2 in co-immunoprecipitation assays. HEK293 cells stably expressing HA-α 2B -AR were transfected with control vector or myc-tagged GGA1 and GGA2. The receptors were immunoprecipitated with α 2B -AR antibodies. The amounts of GGA1 and GGA2 (upper panel) and α 2B -AR (lower panel) were determined by immunoblotting using myc and α 2B -AR antibodies, respectively. Lysate - 1% of total input. Similar results were obtained in three experiments. ( B ) Sequences of the ICL1, ICL2, ICL3 and C-terminus (CT) of α 2B -AR (upper panel) and Coomassie blue staining of purified GST fusion proteins (low panel). The calculated molecular weights of GST and the ICL1, ICL2, ICL3, and CT GST fusion proteins are 27,898, 27,422, 28,070, 43,779 and 29,348 daltons, respectively. ( C ) Interaction of different intracellular domains of α 2B -AR with GGA1 and GGA2. Myc-tagged GGA1 and GGA2 were expressed in HEK293 cells and total cell homogenates were incubated with GST fusion proteins. Bound GGAs were revealed by immunoblotting using anti-myc antibodies. ( D ) Purified His-tagged GGA1 and GGA2. The molecular weight (MW) markers (KDa) are indicated on the left. ( E ) Direct interaction of the α 2B -AR ICL3 with GGA1 and GGA2. Purified His-tagged GGA1 and GGA2 were incubated with GST-ICL3 fusion proteins and bound GGAs were detected by immunoblotting using anti-His antibodies. Similar results were obtained in at least three separate experiments. Lysate −5% of total input. Similar results were obtained in at least 3 experiments.
    Figure Legend Snippet: Interaction of α 2B -AR with GGA1 and GGA2. ( A ) Interaction of α 2B -AR with GGA1 and GGA2 in co-immunoprecipitation assays. HEK293 cells stably expressing HA-α 2B -AR were transfected with control vector or myc-tagged GGA1 and GGA2. The receptors were immunoprecipitated with α 2B -AR antibodies. The amounts of GGA1 and GGA2 (upper panel) and α 2B -AR (lower panel) were determined by immunoblotting using myc and α 2B -AR antibodies, respectively. Lysate - 1% of total input. Similar results were obtained in three experiments. ( B ) Sequences of the ICL1, ICL2, ICL3 and C-terminus (CT) of α 2B -AR (upper panel) and Coomassie blue staining of purified GST fusion proteins (low panel). The calculated molecular weights of GST and the ICL1, ICL2, ICL3, and CT GST fusion proteins are 27,898, 27,422, 28,070, 43,779 and 29,348 daltons, respectively. ( C ) Interaction of different intracellular domains of α 2B -AR with GGA1 and GGA2. Myc-tagged GGA1 and GGA2 were expressed in HEK293 cells and total cell homogenates were incubated with GST fusion proteins. Bound GGAs were revealed by immunoblotting using anti-myc antibodies. ( D ) Purified His-tagged GGA1 and GGA2. The molecular weight (MW) markers (KDa) are indicated on the left. ( E ) Direct interaction of the α 2B -AR ICL3 with GGA1 and GGA2. Purified His-tagged GGA1 and GGA2 were incubated with GST-ICL3 fusion proteins and bound GGAs were detected by immunoblotting using anti-His antibodies. Similar results were obtained in at least three separate experiments. Lysate −5% of total input. Similar results were obtained in at least 3 experiments.

    Techniques Used: Immunoprecipitation, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Staining, Purification, Incubation, Molecular Weight

    Inhibition of cell surface expression of α 2B -AR by shRNA-mediated knockdown of GGA1 and GGA2. ( A ) shRNA-mediated depletion of GGA1 and GGA2 in HEK293 cells. The expression of GGAs was measured by immunoblotting using isoform-specific antibodies. ( B ) Effect of shRNA-mediated knockdown of GGA1 and GGA2 on the cell surface expression of α 2B -AR. HEK293 cells inducibly expressing α 2B -AR were transfected with control or GGA shRNA and then incubated with doxycycline at the concentration of 40 ng/ml for different time periods (0, 4, 8, 12, 16, 20, 24 and 28 h). The cell surface expression of α 2B -AR was determined by intact cell ligand binding using [ 3 H]-RX821002. The data shown are percentages of specific binding obtained from cells transfected with control shRNA and treated with doxycycline for 28 h, in which the mean value of specific [ 3 H]-RX821002 binding was 35,642 ± 985 cpm per well (n = 4) and presented as the mean ± S.E. of at least three individual experiments. * p
    Figure Legend Snippet: Inhibition of cell surface expression of α 2B -AR by shRNA-mediated knockdown of GGA1 and GGA2. ( A ) shRNA-mediated depletion of GGA1 and GGA2 in HEK293 cells. The expression of GGAs was measured by immunoblotting using isoform-specific antibodies. ( B ) Effect of shRNA-mediated knockdown of GGA1 and GGA2 on the cell surface expression of α 2B -AR. HEK293 cells inducibly expressing α 2B -AR were transfected with control or GGA shRNA and then incubated with doxycycline at the concentration of 40 ng/ml for different time periods (0, 4, 8, 12, 16, 20, 24 and 28 h). The cell surface expression of α 2B -AR was determined by intact cell ligand binding using [ 3 H]-RX821002. The data shown are percentages of specific binding obtained from cells transfected with control shRNA and treated with doxycycline for 28 h, in which the mean value of specific [ 3 H]-RX821002 binding was 35,642 ± 985 cpm per well (n = 4) and presented as the mean ± S.E. of at least three individual experiments. * p

    Techniques Used: Inhibition, Expressing, shRNA, Transfection, Incubation, Concentration Assay, Ligand Binding Assay, Binding Assay

    Identification of the GGA1- and GGA2-binding sites in the α 2B -AR ICL3 by progressive deletion. ( A ) Interaction of different ICL3 fragments with the GGA1 hinge and the GGA2 GAE domains. Each ICL3 fragment was generated as GST fusion proteins. The GGA1 hinge and the GGA2 GAE domains were generated as GFP fusion proteins. Their interactions were determined in GST fusion protein pulldown assays. Bound GGA domains were revealed by immunoblotting using GFP antibodies. Bottom panel shows Coomassie blue staining of purified GST fusion proteins. Similar results were obtained in at least three different experiments. The blots from two gels that were run under the same experimental conditions were combined to show the interaction of the GGA1 hinge with different ICL3 domains (upper panel). ( B ) A summary of progressive deletion to identify the GGA1- and GGA2-binding domains in the α 2B -AR ICL3 as shown in ( A ). +Interacting with individual GGA domains; −, not interacting with GGA. ( C ) A diagram showing differential interactions between α 2B -AR and three GGAs. The GGA1 hinge and the GGA2 GAE domains bind to two subdomains of the α 2B -AR ICL3 as revealed in the current studies, whereas the GGA3 VHS domain interacts with the α 2B -AR ICL3, specifically the 3R motif, as demonstrated in our previous studies 47 .
    Figure Legend Snippet: Identification of the GGA1- and GGA2-binding sites in the α 2B -AR ICL3 by progressive deletion. ( A ) Interaction of different ICL3 fragments with the GGA1 hinge and the GGA2 GAE domains. Each ICL3 fragment was generated as GST fusion proteins. The GGA1 hinge and the GGA2 GAE domains were generated as GFP fusion proteins. Their interactions were determined in GST fusion protein pulldown assays. Bound GGA domains were revealed by immunoblotting using GFP antibodies. Bottom panel shows Coomassie blue staining of purified GST fusion proteins. Similar results were obtained in at least three different experiments. The blots from two gels that were run under the same experimental conditions were combined to show the interaction of the GGA1 hinge with different ICL3 domains (upper panel). ( B ) A summary of progressive deletion to identify the GGA1- and GGA2-binding domains in the α 2B -AR ICL3 as shown in ( A ). +Interacting with individual GGA domains; −, not interacting with GGA. ( C ) A diagram showing differential interactions between α 2B -AR and three GGAs. The GGA1 hinge and the GGA2 GAE domains bind to two subdomains of the α 2B -AR ICL3 as revealed in the current studies, whereas the GGA3 VHS domain interacts with the α 2B -AR ICL3, specifically the 3R motif, as demonstrated in our previous studies 47 .

    Techniques Used: Binding Assay, Generated, Staining, Purification

    20) Product Images from "Growth hormone regulates the sensitization of developing peripheral nociceptors during cutaneous inflammation"

    Article Title: Growth hormone regulates the sensitization of developing peripheral nociceptors during cutaneous inflammation

    Journal: Pain

    doi: 10.1097/j.pain.0000000000000770

    Growth hormone (GH) regulates the expression of insulin like growth factor 1 receptor in vitro and after inflammation in vivo at P14 Examples of the single cell collection method used for analysis and examples of primary dorsal root ganglion (DRG) cultures treated with or without GH (A). Single cell PCR results from the various culture conditions show that treatment of primary P14 DRG neurons (n=20) with GH significantly reduces the expression of IGFr1 in single cells (B, C). Example of an amplification plot obtained from a cell treated with GH compared to an untreated DRG neuron shows a rightward shift in the Ct value for IGFr1 in the GH treated neuron while the LYS normalization control gene remained constant in each cell (B). In addition to significantly reduced relative expression, the number of cells that express IGFr1 (IGFR1+) at detectable levels in GH treated cultures was also lower than the number of cells containing IGFr1 in untreated DRG neuron cultures (C). One day (D1) after carrageenan induced inflammation of the hairy hindpaw skin, a significant increase in IGFr1 protein is detected in the DRGs; however this is completely prevented in mice treated with GH at P14 (D; n=3–4 for each age). No changes in the ligand IGF-1 however, were detected in the skin among any of the experimental groups tested (E; n=3–4). Examples of IGFr1 and IGF-1 western blots along with their GAPDH are provided in panels D and E. * p
    Figure Legend Snippet: Growth hormone (GH) regulates the expression of insulin like growth factor 1 receptor in vitro and after inflammation in vivo at P14 Examples of the single cell collection method used for analysis and examples of primary dorsal root ganglion (DRG) cultures treated with or without GH (A). Single cell PCR results from the various culture conditions show that treatment of primary P14 DRG neurons (n=20) with GH significantly reduces the expression of IGFr1 in single cells (B, C). Example of an amplification plot obtained from a cell treated with GH compared to an untreated DRG neuron shows a rightward shift in the Ct value for IGFr1 in the GH treated neuron while the LYS normalization control gene remained constant in each cell (B). In addition to significantly reduced relative expression, the number of cells that express IGFr1 (IGFR1+) at detectable levels in GH treated cultures was also lower than the number of cells containing IGFr1 in untreated DRG neuron cultures (C). One day (D1) after carrageenan induced inflammation of the hairy hindpaw skin, a significant increase in IGFr1 protein is detected in the DRGs; however this is completely prevented in mice treated with GH at P14 (D; n=3–4 for each age). No changes in the ligand IGF-1 however, were detected in the skin among any of the experimental groups tested (E; n=3–4). Examples of IGFr1 and IGF-1 western blots along with their GAPDH are provided in panels D and E. * p

    Techniques Used: Expressing, In Vitro, In Vivo, Polymerase Chain Reaction, Amplification, Mouse Assay, Western Blot

    21) Product Images from "A large-scale functional screen identifies Nova1 and Ncoa3 as regulators of neuronal miRNA function"

    Article Title: A large-scale functional screen identifies Nova1 and Ncoa3 as regulators of neuronal miRNA function

    Journal: The EMBO Journal

    doi: 10.15252/embj.201490643

    Identification of 12 RNA-binding proteins required for miR-134 repressive function in primary neurons using siRNA-based screening
    Figure Legend Snippet: Identification of 12 RNA-binding proteins required for miR-134 repressive function in primary neurons using siRNA-based screening

    Techniques Used: RNA Binding Assay

    22) Product Images from "PED is overexpressed and mediates TRAIL resistance in human non-small cell lung cancer"

    Article Title: PED is overexpressed and mediates TRAIL resistance in human non-small cell lung cancer

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2008.00283.x

    Down-regulation of PED restores TRAIL sensitivity in CALU-1 cells. (A) PED siRNA or a control oligo were transiently transfected in CALU-1 cells in the presence or absence of PED-Myc cDNA. Cells were incubated for 48 or 72 hrs and analysed by Western blotting. The PED siRNA duplex suppressed both exogenous and endogenous PED expression, whereas control siRNA had no effects. (B) PED siRNA effects in A459 and A549 cells. PEDsi RNA, transfected in NSCLC cells was able to reduce PED expression levels (right panel) and induce an increase in TRAIL sensitivity (left panel), as assessed by flow cytometry. Mean ± SD of two independent experiments in duplicate. (C) c-FLIPL siRNA or PED siRNA were transfected as described in Methods. Cells were analysed for c-FLIP expression after 72 hrs incubation. c-FLIPL siRNA but not PED siRNA was able to reduce c-FLIPL expression Effects of silencing PED and c-FLIPL on TRAIL-induced cell death: CALU-1 cells were transfected with siRNA for PED, c-FLIPL or control for 48 hrs, after which cells were trypsinized, plated in 96-well plates in triplicate and further incubated with superkiller TRAIL for 24 hrs. Metabolically active cells were then detected as indicated in the Methods. Mean ± SD of four independent experiments in duplicate. Down-regulation of PED, but not cFLIPL, was responsible for increased sensitivity of CALU-1 cells to TRAIL-mediated cell death.
    Figure Legend Snippet: Down-regulation of PED restores TRAIL sensitivity in CALU-1 cells. (A) PED siRNA or a control oligo were transiently transfected in CALU-1 cells in the presence or absence of PED-Myc cDNA. Cells were incubated for 48 or 72 hrs and analysed by Western blotting. The PED siRNA duplex suppressed both exogenous and endogenous PED expression, whereas control siRNA had no effects. (B) PED siRNA effects in A459 and A549 cells. PEDsi RNA, transfected in NSCLC cells was able to reduce PED expression levels (right panel) and induce an increase in TRAIL sensitivity (left panel), as assessed by flow cytometry. Mean ± SD of two independent experiments in duplicate. (C) c-FLIPL siRNA or PED siRNA were transfected as described in Methods. Cells were analysed for c-FLIP expression after 72 hrs incubation. c-FLIPL siRNA but not PED siRNA was able to reduce c-FLIPL expression Effects of silencing PED and c-FLIPL on TRAIL-induced cell death: CALU-1 cells were transfected with siRNA for PED, c-FLIPL or control for 48 hrs, after which cells were trypsinized, plated in 96-well plates in triplicate and further incubated with superkiller TRAIL for 24 hrs. Metabolically active cells were then detected as indicated in the Methods. Mean ± SD of four independent experiments in duplicate. Down-regulation of PED, but not cFLIPL, was responsible for increased sensitivity of CALU-1 cells to TRAIL-mediated cell death.

    Techniques Used: Transfection, Incubation, Western Blot, Expressing, Flow Cytometry, Cytometry, Metabolic Labelling

    Related Articles

    Transfection:

    Article Title: A quantitative proteomics approach identifies ETV6 and IKZF1 as new regulators of an ERG-driven transcriptional network
    Article Snippet: .. All siRNA (purchased from Qiagen) were labelled with Cy3 using the Silencer siRNA labelling kit (Life Technologies) and 0.3 μM of Cy3-labelled siRNA was used per transfection. .. One hour after electroporation, Cy3-positive cells were isolated using a BD Influx™ cell sorter and allowed to grow for 72 h. RNA was extracted using the RNeasy Mini kit (Qiagen) and complementary DNA synthesis was performed using standard methods as previously described ( ).

    In Vitro:

    Article Title: The Apical Localization of Na+, K+-ATPase in Cultured Human Retinal Pigment Epithelial Cells Depends on Expression of the β2 Subunit
    Article Snippet: .. Reagents and antibodies The following reagents were used: DMEM, F12, PBS, and FBS (GIBCO Cat. 12100-061, Cat. 21700-026, Cat. 21300-058, and Cat. A15-751), the antibiotics penicillin and streptomycin (10,000 U/μg/ml, In vitro , A-01), laminin (SIGMA-ALDRICH Cat. L2020), ITS (a mixture of insulin, human transferrin and selenic acid, BD Biosciences Cat. 354352), Protease Inhibitor Mix (GE Healthcare, Cat. 80-6501-23), a chemiluminescent detection system (ECL Plus; Amersham Biosciences Cat. RPN2132), Lipofectamine 2000 (Invitrogen, Cat. 11668-019), an siRNA Labeling Kit-Cy3 (Ambion by Life Technologies Cat. AM1632), Sp1 siRNA (Sta. .. Cruz Cat. sc-29488), siRNA β1 and β2 (FlexiTube siRNA QIAGEN: SI04284966, SI04249098, SI04173134, SI03149909, SI04273003, SI04138162, SI04274543, SI04284014), the Light Cycler-Fast Start DNAMaster SYBR Green I Kit (Roche, (Applied Biosystems, 4309159), and BCA protein assay reagent (Thermo Scientific, 23224 and 23223).

    Protease Inhibitor:

    Article Title: The Apical Localization of Na+, K+-ATPase in Cultured Human Retinal Pigment Epithelial Cells Depends on Expression of the β2 Subunit
    Article Snippet: .. Reagents and antibodies The following reagents were used: DMEM, F12, PBS, and FBS (GIBCO Cat. 12100-061, Cat. 21700-026, Cat. 21300-058, and Cat. A15-751), the antibiotics penicillin and streptomycin (10,000 U/μg/ml, In vitro , A-01), laminin (SIGMA-ALDRICH Cat. L2020), ITS (a mixture of insulin, human transferrin and selenic acid, BD Biosciences Cat. 354352), Protease Inhibitor Mix (GE Healthcare, Cat. 80-6501-23), a chemiluminescent detection system (ECL Plus; Amersham Biosciences Cat. RPN2132), Lipofectamine 2000 (Invitrogen, Cat. 11668-019), an siRNA Labeling Kit-Cy3 (Ambion by Life Technologies Cat. AM1632), Sp1 siRNA (Sta. .. Cruz Cat. sc-29488), siRNA β1 and β2 (FlexiTube siRNA QIAGEN: SI04284966, SI04249098, SI04173134, SI03149909, SI04273003, SI04138162, SI04274543, SI04284014), the Light Cycler-Fast Start DNAMaster SYBR Green I Kit (Roche, (Applied Biosystems, 4309159), and BCA protein assay reagent (Thermo Scientific, 23224 and 23223).

    Labeling:

    Article Title: An RNA Aptamer Targeting the Receptor Tyrosine Kinase PDGFRα Induces Anti-tumor Effects through STAT3 and p53 in Glioblastoma
    Article Snippet: .. Aptamer RNA was labeled with Cy3 fluorescent dye using the Cy3 Silencer siRNA labeling kit (Thermo Fisher Scientific, Waltham, MA, USA). .. Cy3-labeled aptamers were added to the cells at 200 nM, incubated for 2 hr, and washed for imaging.

    Article Title: The Apical Localization of Na+, K+-ATPase in Cultured Human Retinal Pigment Epithelial Cells Depends on Expression of the β2 Subunit
    Article Snippet: .. Reagents and antibodies The following reagents were used: DMEM, F12, PBS, and FBS (GIBCO Cat. 12100-061, Cat. 21700-026, Cat. 21300-058, and Cat. A15-751), the antibiotics penicillin and streptomycin (10,000 U/μg/ml, In vitro , A-01), laminin (SIGMA-ALDRICH Cat. L2020), ITS (a mixture of insulin, human transferrin and selenic acid, BD Biosciences Cat. 354352), Protease Inhibitor Mix (GE Healthcare, Cat. 80-6501-23), a chemiluminescent detection system (ECL Plus; Amersham Biosciences Cat. RPN2132), Lipofectamine 2000 (Invitrogen, Cat. 11668-019), an siRNA Labeling Kit-Cy3 (Ambion by Life Technologies Cat. AM1632), Sp1 siRNA (Sta. .. Cruz Cat. sc-29488), siRNA β1 and β2 (FlexiTube siRNA QIAGEN: SI04284966, SI04249098, SI04173134, SI03149909, SI04273003, SI04138162, SI04274543, SI04284014), the Light Cycler-Fast Start DNAMaster SYBR Green I Kit (Roche, (Applied Biosystems, 4309159), and BCA protein assay reagent (Thermo Scientific, 23224 and 23223).

    Article Title: Pharmacological Characterization of Chemically Synthesized Monomeric phi29 pRNA Nanoparticles for Systemic Delivery
    Article Snippet: .. The pRNA was labeled with fluoresceine using Silencer ® siRNA labeling kit (Ambion). .. The cells were incubated with the pRNA (200 nM) in absence or presence of excess folate (200 fold) for 30 min., followed by flow analysis ( A-C ), or confocal microscopy ( D-I ).

    Article Title: Valproic Acid Induces Telomerase Reverse Transcriptase Expression during Cortical Development
    Article Snippet: .. Tert siRNA was labeled using Silencer® siRNA Labeling Kit with Cy™3 dye (ThermoFisher Scientific, AM1632) before injection. .. The labeled Tert siRNA was injected into the lateral ventricles of the embryonic brain, and electroporation was conducted by a square wave electroporator (BTX).

    Article Title: Localized glucose and water influx facilitates Cryptosporidium parvum cellular invasion by means of modulation of host-cell membrane protrusion
    Article Snippet: .. The AQP1-siRNA was labeled with Cy3 by using the Silencer siRNA labeling kit (Ambion, Austin, TX). .. Cells were transfected with AQP1-siRNA by using the si-PORT lipid transfection agent (Ambion). pEGFP-N1-AQP1 was generated by inserting the complete rat AQP1 into the expression vector pEGFP-N1 (Invitrogen).

    Article Title: Application of Phi29 Motor pRNA for Targeted Therapeutic Delivery of siRNA Silencing Metallothionein-IIA and Survivin in Ovarian Cancers
    Article Snippet: .. The complementary pRNA/siRNA was stained with Cy3 fluorophores by Silencer siRNA labeling kit (Applied Biosystems) as per manufacturer's instructions prior to cell-binding studies. .. Approximately 700 nmol/l folate-pRNA and control NH2 -pRNA were each incubated with the complementary Cy3-pRNA/siRNA to form dimers.

    Article Title: Nerve Growth Factor Stimulates the Concentration of TrkA within Lipid Rafts and Extracellular Signal-Regulated Kinase Activation through c-Cbl-Associated Protein ▿
    Article Snippet: .. Control and CAP small interfering RNA (siRNA) constructs, a pool of three target-specific 20- to 25-nucleotide siRNA constructs (Santa Cruz Biotechnologies, Santa Cruz, CA) were labeled with carboxyfluorescein (FAM) using a Silencer siRNA labeling kit (Ambion, Austin, TX). .. Dorsal root ganglia (DRG) were isolated from E14 mouse embryos, trypsinized, and transfected with the FAM siRNA-labeled constructs using the Amaxa nucleofector device and the neuron nucleofector kit (Amaxa, Gaithersburg, MD).

    Small Interfering RNA:

    Article Title: Nerve Growth Factor Stimulates the Concentration of TrkA within Lipid Rafts and Extracellular Signal-Regulated Kinase Activation through c-Cbl-Associated Protein ▿
    Article Snippet: .. Control and CAP small interfering RNA (siRNA) constructs, a pool of three target-specific 20- to 25-nucleotide siRNA constructs (Santa Cruz Biotechnologies, Santa Cruz, CA) were labeled with carboxyfluorescein (FAM) using a Silencer siRNA labeling kit (Ambion, Austin, TX). .. Dorsal root ganglia (DRG) were isolated from E14 mouse embryos, trypsinized, and transfected with the FAM siRNA-labeled constructs using the Amaxa nucleofector device and the neuron nucleofector kit (Amaxa, Gaithersburg, MD).

    Construct:

    Article Title: Nerve Growth Factor Stimulates the Concentration of TrkA within Lipid Rafts and Extracellular Signal-Regulated Kinase Activation through c-Cbl-Associated Protein ▿
    Article Snippet: .. Control and CAP small interfering RNA (siRNA) constructs, a pool of three target-specific 20- to 25-nucleotide siRNA constructs (Santa Cruz Biotechnologies, Santa Cruz, CA) were labeled with carboxyfluorescein (FAM) using a Silencer siRNA labeling kit (Ambion, Austin, TX). .. Dorsal root ganglia (DRG) were isolated from E14 mouse embryos, trypsinized, and transfected with the FAM siRNA-labeled constructs using the Amaxa nucleofector device and the neuron nucleofector kit (Amaxa, Gaithersburg, MD).

    Staining:

    Article Title: Application of Phi29 Motor pRNA for Targeted Therapeutic Delivery of siRNA Silencing Metallothionein-IIA and Survivin in Ovarian Cancers
    Article Snippet: .. The complementary pRNA/siRNA was stained with Cy3 fluorophores by Silencer siRNA labeling kit (Applied Biosystems) as per manufacturer's instructions prior to cell-binding studies. .. Approximately 700 nmol/l folate-pRNA and control NH2 -pRNA were each incubated with the complementary Cy3-pRNA/siRNA to form dimers.

    Injection:

    Article Title: Valproic Acid Induces Telomerase Reverse Transcriptase Expression during Cortical Development
    Article Snippet: .. Tert siRNA was labeled using Silencer® siRNA Labeling Kit with Cy™3 dye (ThermoFisher Scientific, AM1632) before injection. .. The labeled Tert siRNA was injected into the lateral ventricles of the embryonic brain, and electroporation was conducted by a square wave electroporator (BTX).

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  • 88
    Thermo Fisher individual sirna duplexes
    The midbody positions the apical surface during cyst development. (A) Caco-2 transfected with control or <t>Cdc42</t> <t>siRNA</t> was fixed and stained for DNA (blue), tubulin (green), and aPKC (red). (top) A control cyst at the two-cell stage (note that abscission appears to have occurred symmetrically). (middle) A larger control cyst, with the midbody in the center of the developing structure (apical region of dividing cell) reflecting asymmetric abscission. (bottom) Cdc42 siRNA structure with one midbody positioned normally at the center and another midbody (located in a different z section) abnormally positioned. (B) Quantitation of midbodies at the center of the cyst from three independent experiments. The total number of midbodies is indicated (N). A midbody is regarded as being in the center if it is located at a distance from the centroid that is less than one third the radius of the structure.
    Individual Sirna Duplexes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 1521 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/individual sirna duplexes/product/Thermo Fisher
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    individual sirna duplexes - by Bioz Stars, 2020-09
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    Thermo Fisher human gnrh r sirna
    Validation of the detection of <t>GnRH-R</t> in immunoblots. A. Representative immunoblots of GnRH-R detection (upper panel) after 48 h transfection with the Human cDNA clone pCMV6-XL5/GNRH-R in 16HBE14o − (1) and CFBE41o − (2) cells. pCMV6-XL5 empty plasmid was used as a control. B. The densitometric analysis after normalization by G3PDH expression and comparison with the controls, indicate that the GnRH-R expression in significantly increased, (n = 5). C. Representative immunoblots of GnRH-R detection after 72 h transfection with a siGENOME individual duplex targeting GnRH-R in 16HBE14o − (1) and CFBE41o − (2) cells. siGENOME Non-Targeting was used as control. A decreased expression of GnRH-R is observed in both cell types. D. The densitometric analysis after normalization by G3PDH expression and comparison with the controls, indicate that the GnRH-R expression is significantly decreased in 16HBE14o − (1) and CFBE41o − (2) cells (n = 7) in the presence of <t>siRNA.</t>
    Human Gnrh R Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The midbody positions the apical surface during cyst development. (A) Caco-2 transfected with control or Cdc42 siRNA was fixed and stained for DNA (blue), tubulin (green), and aPKC (red). (top) A control cyst at the two-cell stage (note that abscission appears to have occurred symmetrically). (middle) A larger control cyst, with the midbody in the center of the developing structure (apical region of dividing cell) reflecting asymmetric abscission. (bottom) Cdc42 siRNA structure with one midbody positioned normally at the center and another midbody (located in a different z section) abnormally positioned. (B) Quantitation of midbodies at the center of the cyst from three independent experiments. The total number of midbodies is indicated (N). A midbody is regarded as being in the center if it is located at a distance from the centroid that is less than one third the radius of the structure.

    Journal: The Journal of Cell Biology

    Article Title: Cdc42 controls spindle orientation to position the apical surface during epithelial morphogenesis

    doi: 10.1083/jcb.200807121

    Figure Lengend Snippet: The midbody positions the apical surface during cyst development. (A) Caco-2 transfected with control or Cdc42 siRNA was fixed and stained for DNA (blue), tubulin (green), and aPKC (red). (top) A control cyst at the two-cell stage (note that abscission appears to have occurred symmetrically). (middle) A larger control cyst, with the midbody in the center of the developing structure (apical region of dividing cell) reflecting asymmetric abscission. (bottom) Cdc42 siRNA structure with one midbody positioned normally at the center and another midbody (located in a different z section) abnormally positioned. (B) Quantitation of midbodies at the center of the cyst from three independent experiments. The total number of midbodies is indicated (N). A midbody is regarded as being in the center if it is located at a distance from the centroid that is less than one third the radius of the structure.

    Article Snippet: RNAi To deplete Cdc42, a SMARTpool (a mixture of four siRNA duplexes) and individual siRNA duplexes were purchased from Thermo Fisher Scientific.

    Techniques: Transfection, Staining, Quantitation Assay

    Cdc42 depletion disrupts mitotic spindle orientation. (A) Diagram depicting spindle angle measurement. The centroid of the cyst (dark blue circle) and the center of the spindle axis (pink circles) of a metaphase cell were drawn using ImageJ. The angle (red) between the spindle axis (black lines) and the line connecting the centroid of the cyst to the center of the spindle (dashed lines) was determined. To analyze spindle poles in different z sections, three z sections were taken so as to include both spindle poles and were merged as shown. Three schematic spindles are shown. The right and middle spindle examples represent correctly oriented spindles whose poles are positioned in one section (z2; middle spindle) or in different sections (z1 and z3; right spindle). The left spindle represents a misoriented spindle whose poles are in different z sections. Spindle microtubules, green; centrosomes, yellow; DNA, light blue. (B) Scatter diagram of metaphase spindle angles in cysts that were transfected with control or two Cdc42 siRNA duplexes from three independent experiments. Pink circles indicate mean values, green circles indicate individual data points, and error bars represent the SEM of the total number of spindles analyzed (N). (C) Caco-2 was transfected with control or Cdc42 siRNA and was fixed and stained for DNA (blue), tubulin (green), and filamentous actin (F-actin; red). Single confocal sections through the center of the cysts are shown. Three z sections are shown for the Cdc42 siRNA cyst to reveal both poles of the misoriented spindle. (D and E) Caco-2 transfected with control or Cdc42 siRNA was fixed and stained for DNA (blue) and aPKC (green). (D) Cdc42 siRNA structures contain cells in the middle, with apical domains present between inner and outer cells. (E) Cdc42 siRNA structures possess apical domains that are not in the center and single cells with more than one apical domain. Arrows indicate cells with multiple distinct apical patches on their surface. Bars, 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: Cdc42 controls spindle orientation to position the apical surface during epithelial morphogenesis

    doi: 10.1083/jcb.200807121

    Figure Lengend Snippet: Cdc42 depletion disrupts mitotic spindle orientation. (A) Diagram depicting spindle angle measurement. The centroid of the cyst (dark blue circle) and the center of the spindle axis (pink circles) of a metaphase cell were drawn using ImageJ. The angle (red) between the spindle axis (black lines) and the line connecting the centroid of the cyst to the center of the spindle (dashed lines) was determined. To analyze spindle poles in different z sections, three z sections were taken so as to include both spindle poles and were merged as shown. Three schematic spindles are shown. The right and middle spindle examples represent correctly oriented spindles whose poles are positioned in one section (z2; middle spindle) or in different sections (z1 and z3; right spindle). The left spindle represents a misoriented spindle whose poles are in different z sections. Spindle microtubules, green; centrosomes, yellow; DNA, light blue. (B) Scatter diagram of metaphase spindle angles in cysts that were transfected with control or two Cdc42 siRNA duplexes from three independent experiments. Pink circles indicate mean values, green circles indicate individual data points, and error bars represent the SEM of the total number of spindles analyzed (N). (C) Caco-2 was transfected with control or Cdc42 siRNA and was fixed and stained for DNA (blue), tubulin (green), and filamentous actin (F-actin; red). Single confocal sections through the center of the cysts are shown. Three z sections are shown for the Cdc42 siRNA cyst to reveal both poles of the misoriented spindle. (D and E) Caco-2 transfected with control or Cdc42 siRNA was fixed and stained for DNA (blue) and aPKC (green). (D) Cdc42 siRNA structures contain cells in the middle, with apical domains present between inner and outer cells. (E) Cdc42 siRNA structures possess apical domains that are not in the center and single cells with more than one apical domain. Arrows indicate cells with multiple distinct apical patches on their surface. Bars, 10 μm.

    Article Snippet: RNAi To deplete Cdc42, a SMARTpool (a mixture of four siRNA duplexes) and individual siRNA duplexes were purchased from Thermo Fisher Scientific.

    Techniques: Transfection, Staining

    Cdc42 depletion induces multiple lumens. (A) Caco-2 was transfected with control or Cdc42 siRNA, plated in three dimensions, and treated with CTX at day 6 to induce luminal swelling. Phase images from cysts before (0 h) and after (12 h) treatment are shown. Note that about half of the Cdc42-depleted cysts lack a single central lumen. Bars, 50 μm. (B) Western blot of Caco-2 transfected with control siRNA, Cdc42 siRNA SMARTpool, or two different individual duplexes. Cdc42 levels are significantly reduced by 3 d and remain reduced for 7 d. (C) Caco-2 cultured as in A was fixed and stained with rhodamine phalloidin. Cysts were examined for single lumen (blue) or multiple lumens (red). The mean ± standard deviation for three independent experiments (at least 50 cysts each) is shown. (D and E) Caco-2 cultured as in A was fixed and stained for DNA (blue) and aPKC (green; D) or DNA (blue), E-cadherin (Ecad; green), and ZO-1 (red; E). Single confocal sections through the middle of the cysts are shown. DIC, differential interference contrast. Bars, 25 μm.

    Journal: The Journal of Cell Biology

    Article Title: Cdc42 controls spindle orientation to position the apical surface during epithelial morphogenesis

    doi: 10.1083/jcb.200807121

    Figure Lengend Snippet: Cdc42 depletion induces multiple lumens. (A) Caco-2 was transfected with control or Cdc42 siRNA, plated in three dimensions, and treated with CTX at day 6 to induce luminal swelling. Phase images from cysts before (0 h) and after (12 h) treatment are shown. Note that about half of the Cdc42-depleted cysts lack a single central lumen. Bars, 50 μm. (B) Western blot of Caco-2 transfected with control siRNA, Cdc42 siRNA SMARTpool, or two different individual duplexes. Cdc42 levels are significantly reduced by 3 d and remain reduced for 7 d. (C) Caco-2 cultured as in A was fixed and stained with rhodamine phalloidin. Cysts were examined for single lumen (blue) or multiple lumens (red). The mean ± standard deviation for three independent experiments (at least 50 cysts each) is shown. (D and E) Caco-2 cultured as in A was fixed and stained for DNA (blue) and aPKC (green; D) or DNA (blue), E-cadherin (Ecad; green), and ZO-1 (red; E). Single confocal sections through the middle of the cysts are shown. DIC, differential interference contrast. Bars, 25 μm.

    Article Snippet: RNAi To deplete Cdc42, a SMARTpool (a mixture of four siRNA duplexes) and individual siRNA duplexes were purchased from Thermo Fisher Scientific.

    Techniques: Transfection, Western Blot, Cell Culture, Staining, Standard Deviation

    Downregulation of Aip1 expression increases paracellular permeability of colonic epithelial cell monolayers. SK-CO15 epithelial cells were transfected with different Aip1-specific siRNA duplexes and corresponding control siRNAs. On day 4 posttransfection, the efficiency of siRNA knockdown as well as the expression of Aip1-interacting cytoskeletal regulators and apoptosis markers was examined by electrophoresis and immunoblotting ( A ). ADF, actin-depolymerizing factor; PARP, poly(ADP-ribose) polymerase. Permeability of control and Aip1-depleted SK-CO15 cells was examined by measuring transepithelial electrical resistance (TEER) ( B ) and transepithelial flux of fluoresceinated dextrans ( C ). Data are presented as means ± SE ( n = 3); ** P

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: Actin-interacting protein 1 controls assembly and permeability of intestinal epithelial apical junctions

    doi: 10.1152/ajpgi.00446.2014

    Figure Lengend Snippet: Downregulation of Aip1 expression increases paracellular permeability of colonic epithelial cell monolayers. SK-CO15 epithelial cells were transfected with different Aip1-specific siRNA duplexes and corresponding control siRNAs. On day 4 posttransfection, the efficiency of siRNA knockdown as well as the expression of Aip1-interacting cytoskeletal regulators and apoptosis markers was examined by electrophoresis and immunoblotting ( A ). ADF, actin-depolymerizing factor; PARP, poly(ADP-ribose) polymerase. Permeability of control and Aip1-depleted SK-CO15 cells was examined by measuring transepithelial electrical resistance (TEER) ( B ) and transepithelial flux of fluoresceinated dextrans ( C ). Data are presented as means ± SE ( n = 3); ** P

    Article Snippet: Small-interference RNA (siRNA)-mediated knockdown of Aip1 was carried out using individual siRNA duplexes obtained either from Dharmacon-Thermo Scientific (Waltham, MA) or Qiagen (Venlo, Limburg), as previously described ( , ).

    Techniques: Expressing, Permeability, Transfection, Electrophoresis

    Incubation with Mithramycin A and siRNA knockdown of Sp1 and Sp3 inhibit basal RIIα protein expression in human myometrial cells. (A) Decrease in RIIα protein levels monitored by Western blotting after 6 hrs treatment of human myometrial cells with 2 μM Mithramycin. Data are expressed as mean ± S.E.M. Data were obtained from six myometrial cell preparations. *P, 0.05 Student’s t-test (n 5 6) treated versus untreated cells. (B) Mithramycin A at 200 νM also decreased RIIα protein levels as monitored by Western blotting after 24 hrs treatment of human myometrial cells indicating further specificity of the effect. Data are expressed as mean ± S.E.M. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Epigenetic modulation of the protein kinase A RIIα (PRKAR2A) gene by histone deacetylases 1 and 2 in human smooth muscle cells

    doi: 10.1111/j.1582-4934.2009.00927.x

    Figure Lengend Snippet: Incubation with Mithramycin A and siRNA knockdown of Sp1 and Sp3 inhibit basal RIIα protein expression in human myometrial cells. (A) Decrease in RIIα protein levels monitored by Western blotting after 6 hrs treatment of human myometrial cells with 2 μM Mithramycin. Data are expressed as mean ± S.E.M. Data were obtained from six myometrial cell preparations. *P, 0.05 Student’s t-test (n 5 6) treated versus untreated cells. (B) Mithramycin A at 200 νM also decreased RIIα protein levels as monitored by Western blotting after 24 hrs treatment of human myometrial cells indicating further specificity of the effect. Data are expressed as mean ± S.E.M. * P

    Article Snippet: Dharmacon siGENOME™ SMARTpools (Thermo Fisher Scientific, Lafayette, CO, USA) of four specific siRNA duplexes (targeted to individual Sp1 and Sp3 species, 100 nM siRNA each) were used to transfect myometrial cells using the Dharmafect™ 1 lipid reagent (Thermo Fisher).

    Techniques: Incubation, Expressing, Western Blot

    Validation of the detection of GnRH-R in immunoblots. A. Representative immunoblots of GnRH-R detection (upper panel) after 48 h transfection with the Human cDNA clone pCMV6-XL5/GNRH-R in 16HBE14o − (1) and CFBE41o − (2) cells. pCMV6-XL5 empty plasmid was used as a control. B. The densitometric analysis after normalization by G3PDH expression and comparison with the controls, indicate that the GnRH-R expression in significantly increased, (n = 5). C. Representative immunoblots of GnRH-R detection after 72 h transfection with a siGENOME individual duplex targeting GnRH-R in 16HBE14o − (1) and CFBE41o − (2) cells. siGENOME Non-Targeting was used as control. A decreased expression of GnRH-R is observed in both cell types. D. The densitometric analysis after normalization by G3PDH expression and comparison with the controls, indicate that the GnRH-R expression is significantly decreased in 16HBE14o − (1) and CFBE41o − (2) cells (n = 7) in the presence of siRNA.

    Journal: PLoS ONE

    Article Title: Improvement of Chloride Transport Defect by Gonadotropin-Releasing Hormone (GnRH) in Cystic Fibrosis Epithelial Cells

    doi: 10.1371/journal.pone.0088964

    Figure Lengend Snippet: Validation of the detection of GnRH-R in immunoblots. A. Representative immunoblots of GnRH-R detection (upper panel) after 48 h transfection with the Human cDNA clone pCMV6-XL5/GNRH-R in 16HBE14o − (1) and CFBE41o − (2) cells. pCMV6-XL5 empty plasmid was used as a control. B. The densitometric analysis after normalization by G3PDH expression and comparison with the controls, indicate that the GnRH-R expression in significantly increased, (n = 5). C. Representative immunoblots of GnRH-R detection after 72 h transfection with a siGENOME individual duplex targeting GnRH-R in 16HBE14o − (1) and CFBE41o − (2) cells. siGENOME Non-Targeting was used as control. A decreased expression of GnRH-R is observed in both cell types. D. The densitometric analysis after normalization by G3PDH expression and comparison with the controls, indicate that the GnRH-R expression is significantly decreased in 16HBE14o − (1) and CFBE41o − (2) cells (n = 7) in the presence of siRNA.

    Article Snippet: Transfection For GnRH-R overexpression and inhibition, cells were transfected using Lipofectamine 2000 (Life Technologies Corporation, Carlsbad, CA, USA), according to the manufacturer’s instructions, with either the human cDNA clone pCMV6-XL5/GNRH-R (OriGene Technologies Inc., Rockville, MD, USA) or the human GnRH-R siRNA (5′-GGAAUUUGGUAUUGGUUUG-3′, siGENOME individual duplex (Thermo Fisher Scientific Inc., Waltham, MA, USA).

    Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing