hek293 cells  (Thermo Fisher)


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    Structured Review

    Thermo Fisher hek293 cells
    The impact of SNP rs2273626 in the seed sequence of miR-4707 on miRNA production and targeting. (A) The figure shows the predicted hairpin structure of miR-4707 containing rs2273626, which was associated with VCDR and cup area. The mature miRNA sequences (3p and 5p) are shown in red and the position of variants is depicted by an arrow. To examine the effect of rs2273626 on the miR-4707 expression level, <t>HEK293</t> cells were transfected with GFP-miRNA transcripts containing either the minor allele T or the major allele G. The levels of mature miRNA relative to GFP transcript levels were calculated. (B) Luciferase reporter assays indicating miR-4707-3p–mediated repression of CARD10. HEK293 cells were cotransfected with CARD10 3′UTR luciferase reporter vector and GFP-miRNA transcripts containing either the minor allele T or the major allele G. This experiment indicates a significant difference (P = 0.04) between the relative luciferase activity of the CARD10 3′UTR construct in the presence of miR-4707-3p containing the major allele and the minor allele. Our results suggest that rs2273626 diminishes the regulatory interaction between miR-4707-3p and CARD10, resulting in increased CARD10 levels. All experiments were performed in triplicates and repeated at least three times. Error bars represent standard deviation (SD). NS, nonsignificant.
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    Images

    1) Product Images from "A Genome-Wide Scan for MicroRNA-Related Genetic Variants Associated With Primary Open-Angle Glaucoma"

    Article Title: A Genome-Wide Scan for MicroRNA-Related Genetic Variants Associated With Primary Open-Angle Glaucoma

    Journal: Investigative Ophthalmology & Visual Science

    doi: 10.1167/iovs.17-22410

    The impact of SNP rs2273626 in the seed sequence of miR-4707 on miRNA production and targeting. (A) The figure shows the predicted hairpin structure of miR-4707 containing rs2273626, which was associated with VCDR and cup area. The mature miRNA sequences (3p and 5p) are shown in red and the position of variants is depicted by an arrow. To examine the effect of rs2273626 on the miR-4707 expression level, HEK293 cells were transfected with GFP-miRNA transcripts containing either the minor allele T or the major allele G. The levels of mature miRNA relative to GFP transcript levels were calculated. (B) Luciferase reporter assays indicating miR-4707-3p–mediated repression of CARD10. HEK293 cells were cotransfected with CARD10 3′UTR luciferase reporter vector and GFP-miRNA transcripts containing either the minor allele T or the major allele G. This experiment indicates a significant difference (P = 0.04) between the relative luciferase activity of the CARD10 3′UTR construct in the presence of miR-4707-3p containing the major allele and the minor allele. Our results suggest that rs2273626 diminishes the regulatory interaction between miR-4707-3p and CARD10, resulting in increased CARD10 levels. All experiments were performed in triplicates and repeated at least three times. Error bars represent standard deviation (SD). NS, nonsignificant.
    Figure Legend Snippet: The impact of SNP rs2273626 in the seed sequence of miR-4707 on miRNA production and targeting. (A) The figure shows the predicted hairpin structure of miR-4707 containing rs2273626, which was associated with VCDR and cup area. The mature miRNA sequences (3p and 5p) are shown in red and the position of variants is depicted by an arrow. To examine the effect of rs2273626 on the miR-4707 expression level, HEK293 cells were transfected with GFP-miRNA transcripts containing either the minor allele T or the major allele G. The levels of mature miRNA relative to GFP transcript levels were calculated. (B) Luciferase reporter assays indicating miR-4707-3p–mediated repression of CARD10. HEK293 cells were cotransfected with CARD10 3′UTR luciferase reporter vector and GFP-miRNA transcripts containing either the minor allele T or the major allele G. This experiment indicates a significant difference (P = 0.04) between the relative luciferase activity of the CARD10 3′UTR construct in the presence of miR-4707-3p containing the major allele and the minor allele. Our results suggest that rs2273626 diminishes the regulatory interaction between miR-4707-3p and CARD10, resulting in increased CARD10 levels. All experiments were performed in triplicates and repeated at least three times. Error bars represent standard deviation (SD). NS, nonsignificant.

    Techniques Used: Sequencing, Expressing, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Construct, Standard Deviation

    2) Product Images from "Lamin B is a target for selective nuclear PQC by BAG3: implication for nuclear envelopathies"

    Article Title: Lamin B is a target for selective nuclear PQC by BAG3: implication for nuclear envelopathies

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1255-9

    BAG3 interacts with nuclear protein lamin B using BAG domain. a Co-immunoprecipitation studies show that HSP70 co-immunoprecipitated with BAG3. HEK293 cells were transfected with BAG3 plasmid for 48 h and immunoprecipitation was done with FLAG-tag antibody. Western blot was done with HSP70 antibody. b , c Immunoprecipitation study shows that BAG3 can interact with nuclear envelop protein lamin B but not with lamin A/C. HEK293 cells were co-transfected with BAG3 and lamin B-mCherry or BAG3 and lamin A/C-mCherry. Immunoprecipitation was done with the FLAG antibody and western blots were done using lamin B or lamin A/C antibody, respectively. d Western blot shows that BAG3 interacts with the ubiquitin. HEK293 cells were co-transfected with BAG3 and ubiquitin expressor plasmids. Immunoprecipitation was done with FLAG antibody and western blots were done with ubiquitin antibody. e Schematic diagram shows different domains of BAG3. f Western blot analysis showing detection of full-length BAG3 and the BAG3 deletion mutants by anti-FLAG. g–i Co-immunoprecipitation of BAG3 (using anti-FLAG antibody) followed by western blot for detection of lamin B as well as the BAG3 partners, HSP70 and DNAJ, respectively. j Representative images show that BAG domain can restore the wild-type phenotype in the CRISPR-mediated BAG3 mutant C2C12 cells. Wild-type and mutant C2C12 cells were transfected with the mutant and wild-type BAG3 gene using Lipofectamine for 48 h. Cells were fixed with 4% PFA and immunocytochemistry was done with the FLAG-tag antibody (green) and nuclei were stained with DAPI (blue). Images were captured with confocal microscopy. k Quantification of the results (shown in j ) in which nuclear area was measured using the ImageJ software ( n = 100 cells, in each group)
    Figure Legend Snippet: BAG3 interacts with nuclear protein lamin B using BAG domain. a Co-immunoprecipitation studies show that HSP70 co-immunoprecipitated with BAG3. HEK293 cells were transfected with BAG3 plasmid for 48 h and immunoprecipitation was done with FLAG-tag antibody. Western blot was done with HSP70 antibody. b , c Immunoprecipitation study shows that BAG3 can interact with nuclear envelop protein lamin B but not with lamin A/C. HEK293 cells were co-transfected with BAG3 and lamin B-mCherry or BAG3 and lamin A/C-mCherry. Immunoprecipitation was done with the FLAG antibody and western blots were done using lamin B or lamin A/C antibody, respectively. d Western blot shows that BAG3 interacts with the ubiquitin. HEK293 cells were co-transfected with BAG3 and ubiquitin expressor plasmids. Immunoprecipitation was done with FLAG antibody and western blots were done with ubiquitin antibody. e Schematic diagram shows different domains of BAG3. f Western blot analysis showing detection of full-length BAG3 and the BAG3 deletion mutants by anti-FLAG. g–i Co-immunoprecipitation of BAG3 (using anti-FLAG antibody) followed by western blot for detection of lamin B as well as the BAG3 partners, HSP70 and DNAJ, respectively. j Representative images show that BAG domain can restore the wild-type phenotype in the CRISPR-mediated BAG3 mutant C2C12 cells. Wild-type and mutant C2C12 cells were transfected with the mutant and wild-type BAG3 gene using Lipofectamine for 48 h. Cells were fixed with 4% PFA and immunocytochemistry was done with the FLAG-tag antibody (green) and nuclei were stained with DAPI (blue). Images were captured with confocal microscopy. k Quantification of the results (shown in j ) in which nuclear area was measured using the ImageJ software ( n = 100 cells, in each group)

    Techniques Used: Immunoprecipitation, Transfection, Plasmid Preparation, FLAG-tag, Western Blot, CRISPR, Mutagenesis, Immunocytochemistry, Staining, Confocal Microscopy, Software

    3) Product Images from "Expression of N-terminal truncated desmoglein 3 (?NDg3) in epidermis and its role in keratinocyte differentiation"

    Article Title: Expression of N-terminal truncated desmoglein 3 (?NDg3) in epidermis and its role in keratinocyte differentiation

    Journal: Experimental & Molecular Medicine

    doi: 10.3858/emm.2009.41.1.006

    (A) RT-PCR analysis. Primary normal human epidermal keratinocytes were treated with high calcium at the indicated time points. Two µg of total RNAs were reverse transcribed with M-MLV reverse transcriptase and used for PCR amplification. (B) Western blot analysis. Cellular proteins were extracted from primary cultured keratinocytes, then separated on polyacrylamide gels. Blot was probed with C-term anti-Dg3 antibody. The positive control (P) for 31 kDa protein was prepared by transfection of pcDNA3.1-ΔNDg3 to HEK293 cells.
    Figure Legend Snippet: (A) RT-PCR analysis. Primary normal human epidermal keratinocytes were treated with high calcium at the indicated time points. Two µg of total RNAs were reverse transcribed with M-MLV reverse transcriptase and used for PCR amplification. (B) Western blot analysis. Cellular proteins were extracted from primary cultured keratinocytes, then separated on polyacrylamide gels. Blot was probed with C-term anti-Dg3 antibody. The positive control (P) for 31 kDa protein was prepared by transfection of pcDNA3.1-ΔNDg3 to HEK293 cells.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Western Blot, Cell Culture, Positive Control, Transfection

    4) Product Images from "Comparative Genomic and Sequence Analysis Provides Insight into the Molecular Functionality of NOD1 and NOD2"

    Article Title: Comparative Genomic and Sequence Analysis Provides Insight into the Molecular Functionality of NOD1 and NOD2

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2013.00317

    The impact of mutation of conserved residues between the C-terminus of human NOD1 and NOD2 on receptor function . (A) Alignment of the terminal 33 amino acids of human NOD1 and NOD2. Residues highlighted in cyan are conserved across mammals in the relevant protein. The consensus sequence highlights residues found in the termini of both human NOD1 and NOD2. (B) NFκB luciferase reporter assays were performed in HEK293 cells using wild-type pCMV-NOD2 and the point mutants E1026A, E1027A, and R1037A. DNA (0.1 ng/well) was transfected into 96-well plates with (black bars) and without (white bars) muramyl dipeptide (MDP). After 24 h cells were lysed and NFκB activity determined. Results show the average of three independent experiments and * p
    Figure Legend Snippet: The impact of mutation of conserved residues between the C-terminus of human NOD1 and NOD2 on receptor function . (A) Alignment of the terminal 33 amino acids of human NOD1 and NOD2. Residues highlighted in cyan are conserved across mammals in the relevant protein. The consensus sequence highlights residues found in the termini of both human NOD1 and NOD2. (B) NFκB luciferase reporter assays were performed in HEK293 cells using wild-type pCMV-NOD2 and the point mutants E1026A, E1027A, and R1037A. DNA (0.1 ng/well) was transfected into 96-well plates with (black bars) and without (white bars) muramyl dipeptide (MDP). After 24 h cells were lysed and NFκB activity determined. Results show the average of three independent experiments and * p

    Techniques Used: Mutagenesis, Sequencing, Luciferase, Transfection, Activity Assay

    Amino acid conservation in the NOD1 and NOD2 CARDs . Cartoon and surface representations of NOD1 CARD (A) , NOD2 CARD1 (C) , and NOD2 CARD2 (D) showing amino acids conserved across all species (green) and conserved across mammals (pink). In each panel the top and bottom images are related by a 180° rotation around the vertical axis. The left and right images are cartoon and surface representations of the same view respectively. Residues previously implicated in interaction with RIP2 (NOD1 – E53, D54, E56; NOD2 – R38, R36) or in the process of ubiquitination (NOD1 – E84, Y88; NOD2 – I104, L200) are labeled and presented as spheres. Conservation is mapped onto an experimental NOD1 structure (PDB ID: 2DBD) and homology models of the NOD2 CARDs. (B) Differential contributions to receptor activation. NFκB luciferase reporter assays were performed in HEK293 cells using wild-type (WT) NOD1, E53A, D54A, and E56A constructs. DNA (0.1 ng/well) and varying concentrations of stimulatory (i.e., DAP) or control (i.e., Lys) ligands were transfected into 96-well plates. After 24 h cells were lysed and NFκB activity determined. Results show the average of four independent experiments and ** p
    Figure Legend Snippet: Amino acid conservation in the NOD1 and NOD2 CARDs . Cartoon and surface representations of NOD1 CARD (A) , NOD2 CARD1 (C) , and NOD2 CARD2 (D) showing amino acids conserved across all species (green) and conserved across mammals (pink). In each panel the top and bottom images are related by a 180° rotation around the vertical axis. The left and right images are cartoon and surface representations of the same view respectively. Residues previously implicated in interaction with RIP2 (NOD1 – E53, D54, E56; NOD2 – R38, R36) or in the process of ubiquitination (NOD1 – E84, Y88; NOD2 – I104, L200) are labeled and presented as spheres. Conservation is mapped onto an experimental NOD1 structure (PDB ID: 2DBD) and homology models of the NOD2 CARDs. (B) Differential contributions to receptor activation. NFκB luciferase reporter assays were performed in HEK293 cells using wild-type (WT) NOD1, E53A, D54A, and E56A constructs. DNA (0.1 ng/well) and varying concentrations of stimulatory (i.e., DAP) or control (i.e., Lys) ligands were transfected into 96-well plates. After 24 h cells were lysed and NFκB activity determined. Results show the average of four independent experiments and ** p

    Techniques Used: Labeling, Activation Assay, Luciferase, Construct, Transfection, Activity Assay

    5) Product Images from "Toxoplasma gondii cathepsin proteases are undeveloped prominent vaccine antigens against toxoplasmosis"

    Article Title: Toxoplasma gondii cathepsin proteases are undeveloped prominent vaccine antigens against toxoplasmosis

    Journal: BMC Infectious Diseases

    doi: 10.1186/1471-2334-13-207

    Indirect fluorescent antibody detection of recombinant TgCPB and TgCPL proteases on the surface of HEK293 cells. ( A1 ) pTgCPB-transfected HEK293 cells; ( A2 ) pBudCE4.1-transfected HEK293 cells. ( B1 ) pTgCPL-transfected HEK293 cells; ( B2 ) pBudCE4.1-transfected HEK293 cells. ( C1 ) pTgCPB/TgCPL-transfected HEK293 cells where pTgCPB/TgCPL expression was detected using the anti-TgCPB antibody as the primary antibody; ( C2 ) pBudCE4.1-transfected HEK293 cells where the anti-TgCPB antibody was used as the primary antibody. ( D1 ) pTgCPB/TgCPL-transfected HEK293 cells where the anti-TgCPL antibody was used as the primary antibody; ( D2 ) pBudCE4.1-transfected HEK293 cells where the anti-TgCPL antibody was used as the primary antibody. High level of laser intensity was used for A1, A2, B1 and B2, lower level of laser intensity for C1, C2, D1 and D2.
    Figure Legend Snippet: Indirect fluorescent antibody detection of recombinant TgCPB and TgCPL proteases on the surface of HEK293 cells. ( A1 ) pTgCPB-transfected HEK293 cells; ( A2 ) pBudCE4.1-transfected HEK293 cells. ( B1 ) pTgCPL-transfected HEK293 cells; ( B2 ) pBudCE4.1-transfected HEK293 cells. ( C1 ) pTgCPB/TgCPL-transfected HEK293 cells where pTgCPB/TgCPL expression was detected using the anti-TgCPB antibody as the primary antibody; ( C2 ) pBudCE4.1-transfected HEK293 cells where the anti-TgCPB antibody was used as the primary antibody. ( D1 ) pTgCPB/TgCPL-transfected HEK293 cells where the anti-TgCPL antibody was used as the primary antibody; ( D2 ) pBudCE4.1-transfected HEK293 cells where the anti-TgCPL antibody was used as the primary antibody. High level of laser intensity was used for A1, A2, B1 and B2, lower level of laser intensity for C1, C2, D1 and D2.

    Techniques Used: Recombinant, Transfection, Expressing

    Western blot analysis of TgCPB and TgCPL protein expression in transfected HEK293 cells. M: protein marker; (1) HEK293 cells transfected with the recombinant pTgCPB/TgCPL plasmid; (2) HEK293 cells transfected with the recombinant pTgCPL plasmid; (3) HEK293 cells transfected with the recombinant pTgCPB plasmid; (4) HEK293 cells transfected with an empty vector.
    Figure Legend Snippet: Western blot analysis of TgCPB and TgCPL protein expression in transfected HEK293 cells. M: protein marker; (1) HEK293 cells transfected with the recombinant pTgCPB/TgCPL plasmid; (2) HEK293 cells transfected with the recombinant pTgCPL plasmid; (3) HEK293 cells transfected with the recombinant pTgCPB plasmid; (4) HEK293 cells transfected with an empty vector.

    Techniques Used: Western Blot, Expressing, Transfection, Marker, Recombinant, Plasmid Preparation

    6) Product Images from "LRRK2 functions as a Wnt signaling scaffold, bridging cytosolic proteins and membrane-localized LRP6"

    Article Title: LRRK2 functions as a Wnt signaling scaffold, bridging cytosolic proteins and membrane-localized LRP6

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/dds342

    LRRK2 binds directly to the intracellular domain of LRP6. ( A ) Myc-tagged LRRK2 (red) and HA-tagged LRP6 (green) show almost complete co-localization in HEK293 cells (a–g). Almost no co-localization was seen between FLAG-tagged DVL1 (red) and HA-tagged LRP6 (green) (h–n). ( B ) FLAG-tagged LRRK2 and HA-tagged LRP6 co-immunoprecipitate from HEK293 cells. ( C ) The intracellular domain of LRP6 binds the LRRK2-RocCOR domain but not DVL2 in yeast. X-gal freeze-fracture assays indicate protein–protein interactions in blue. All negative controls show no color change. ( D ) Consistent with a requirement for the LRRK2-RocCOR domain for the LRP6–LRRK2 interaction the myc-tagged LRRK2-RocCOR domain (green) co-localizes with HA-tagged LRP6 similar to full-length protein in HEK293 cells (A, a–g). In (A) and (D), DNA staining with 4’,6-diamidino-2-phenylindole (blue) is shown, scale bar: 10 μm.
    Figure Legend Snippet: LRRK2 binds directly to the intracellular domain of LRP6. ( A ) Myc-tagged LRRK2 (red) and HA-tagged LRP6 (green) show almost complete co-localization in HEK293 cells (a–g). Almost no co-localization was seen between FLAG-tagged DVL1 (red) and HA-tagged LRP6 (green) (h–n). ( B ) FLAG-tagged LRRK2 and HA-tagged LRP6 co-immunoprecipitate from HEK293 cells. ( C ) The intracellular domain of LRP6 binds the LRRK2-RocCOR domain but not DVL2 in yeast. X-gal freeze-fracture assays indicate protein–protein interactions in blue. All negative controls show no color change. ( D ) Consistent with a requirement for the LRRK2-RocCOR domain for the LRP6–LRRK2 interaction the myc-tagged LRRK2-RocCOR domain (green) co-localizes with HA-tagged LRP6 similar to full-length protein in HEK293 cells (A, a–g). In (A) and (D), DNA staining with 4’,6-diamidino-2-phenylindole (blue) is shown, scale bar: 10 μm.

    Techniques Used: Staining

    LRRK2 associates with the BDC and DVL proteins. ( A ) Overview of canonical Wnt signaling and potential interactions with LRRK2. ( B ) LRRK2 co-immunoprecipitates from mouse brain cytoplasm with components of the BDC and DVL proteins. Immunoprecipitations with anti-LRRK2 antibody (MJFF2). Anti-LRRK2 IP confirmed by western blotting with a second anti-LRRK2 antibody (NeuroMab N138/6). LRRK2 and co-complexed proteins are present in MJFF2 eluates and cell lysate, but not IgG control. ( C ) siRNA-mediated knockdown of LRRK2 increased basal and Wnt3a-induced TOPflash activity in SH-SY5Y cells. For each treatment condition, values are normalized to control siRNA to show the effect of LRRK2 knockdown. siRNA to Axin1 used as a positive control. P -values relative to siRNA control are shown. ( D ) Knockdown of LRRK1 and/or LRRK2 enhances DVL1-mediated Wnt signaling in HEK293 cells. P -values relative to siRNA control are shown.
    Figure Legend Snippet: LRRK2 associates with the BDC and DVL proteins. ( A ) Overview of canonical Wnt signaling and potential interactions with LRRK2. ( B ) LRRK2 co-immunoprecipitates from mouse brain cytoplasm with components of the BDC and DVL proteins. Immunoprecipitations with anti-LRRK2 antibody (MJFF2). Anti-LRRK2 IP confirmed by western blotting with a second anti-LRRK2 antibody (NeuroMab N138/6). LRRK2 and co-complexed proteins are present in MJFF2 eluates and cell lysate, but not IgG control. ( C ) siRNA-mediated knockdown of LRRK2 increased basal and Wnt3a-induced TOPflash activity in SH-SY5Y cells. For each treatment condition, values are normalized to control siRNA to show the effect of LRRK2 knockdown. siRNA to Axin1 used as a positive control. P -values relative to siRNA control are shown. ( D ) Knockdown of LRRK1 and/or LRRK2 enhances DVL1-mediated Wnt signaling in HEK293 cells. P -values relative to siRNA control are shown.

    Techniques Used: Western Blot, Activity Assay, Positive Control

    LRRK2 is recruited to membranes by Wnt3a. Acute treatment of HEK293 cells with recombinant Wnt3a increases the amount of endogenous LRRK2 present in crude membrane fractions. ( A ) A representative experiment. ( B and C ) Quantifications of the relative levels of membrane-associated LRRK2 at each time point from four independent experiments, normalized to (B) calnexin and (C) Rab5b. ( D ) Confocal images showing HEK293 cells expressing LRRK2 under basal conditions (A–D) and after activation with Wnt3a ( E – H ), scale bar: 10 μm.
    Figure Legend Snippet: LRRK2 is recruited to membranes by Wnt3a. Acute treatment of HEK293 cells with recombinant Wnt3a increases the amount of endogenous LRRK2 present in crude membrane fractions. ( A ) A representative experiment. ( B and C ) Quantifications of the relative levels of membrane-associated LRRK2 at each time point from four independent experiments, normalized to (B) calnexin and (C) Rab5b. ( D ) Confocal images showing HEK293 cells expressing LRRK2 under basal conditions (A–D) and after activation with Wnt3a ( E – H ), scale bar: 10 μm.

    Techniques Used: Recombinant, Expressing, Activation Assay

    7) Product Images from "Membrane Transporters for Sulfated Steroids in the Human Testis - Cellular Localization, Expression Pattern and Functional Analysis"

    Article Title: Membrane Transporters for Sulfated Steroids in the Human Testis - Cellular Localization, Expression Pattern and Functional Analysis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0062638

    Transport studies with sulfated steroid hormones. ( A ) SOAT-HEK293 were incubated with 10 µM non-radiolabeled estrone-3-sulfate, DHEAS, β-estradiol-3-sulfate, and androstenediol-3-sulfate in the presence (black bars) or absence (open bars) of sodium over 10 min at 37°C. Cells lysates were analyzed by LC-MS-MS in order to determine the absolute cell-associated amount of the steroid sulfate molecules in their intact forms. ( B ) Transport studies with 10 µM [ 3 H]estrone-3-sulfate on stably transfected SOAT-, OATP6A1-, OATP1C1-, and OSCP1-HEK293 cells in the presence and absence of sodium. Non-transfected HEK293 cells served as a control. In contrast to SOAT, OATP6A1, OATP1C1, and OSCP1 showed no significant transport function for E1S.
    Figure Legend Snippet: Transport studies with sulfated steroid hormones. ( A ) SOAT-HEK293 were incubated with 10 µM non-radiolabeled estrone-3-sulfate, DHEAS, β-estradiol-3-sulfate, and androstenediol-3-sulfate in the presence (black bars) or absence (open bars) of sodium over 10 min at 37°C. Cells lysates were analyzed by LC-MS-MS in order to determine the absolute cell-associated amount of the steroid sulfate molecules in their intact forms. ( B ) Transport studies with 10 µM [ 3 H]estrone-3-sulfate on stably transfected SOAT-, OATP6A1-, OATP1C1-, and OSCP1-HEK293 cells in the presence and absence of sodium. Non-transfected HEK293 cells served as a control. In contrast to SOAT, OATP6A1, OATP1C1, and OSCP1 showed no significant transport function for E1S.

    Techniques Used: Incubation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Stable Transfection, Transfection

    SOAT expression in stably transfected SOAT-HEK293 cells. In the SOAT-HEK293 cells SOAT expression was induced by pre-treatment with tetracycline (+Tet). Control cells were untreated with tetracycline (−Tet) or represent non-transfected HEK293 cells (contr.). ( A ) Cell lysates were processed for WB analysis with the SOAT 311–377 antibody and revealed an apparent molecular weight of 49–55 kDa, likely representing different glycosylation states of the SOAT protein. ( B ) SOAT expression was directed to the plasma membrane of HEK293 cells by immunofluorescence analysis with the SOAT 311–377 antibody (green fluorescence). Nuclear staining with DAPI (blue fluorescence). Scale bar: 25 µm.
    Figure Legend Snippet: SOAT expression in stably transfected SOAT-HEK293 cells. In the SOAT-HEK293 cells SOAT expression was induced by pre-treatment with tetracycline (+Tet). Control cells were untreated with tetracycline (−Tet) or represent non-transfected HEK293 cells (contr.). ( A ) Cell lysates were processed for WB analysis with the SOAT 311–377 antibody and revealed an apparent molecular weight of 49–55 kDa, likely representing different glycosylation states of the SOAT protein. ( B ) SOAT expression was directed to the plasma membrane of HEK293 cells by immunofluorescence analysis with the SOAT 311–377 antibody (green fluorescence). Nuclear staining with DAPI (blue fluorescence). Scale bar: 25 µm.

    Techniques Used: Expressing, Stable Transfection, Transfection, Western Blot, Molecular Weight, Immunofluorescence, Fluorescence, Staining

    8) Product Images from "Regulation of ABCC6 Trafficking and Stability by a Conserved C-terminal PDZ-Like Sequence"

    Article Title: Regulation of ABCC6 Trafficking and Stability by a Conserved C-terminal PDZ-Like Sequence

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0097360

    Alteration of ABCC6 trafficking by the PDZ-like C-terminus. To evaluate the potential role of the PDZ-like sequences at the C-terminus of ABCC6, wildtype and mutant proteins were expressed in HEK293 cells and evaluated by western blotting and immunofluorescence. A, a cartoon illustrating the domain organization and topology of ABCC6 is shown. ABCC6 contains three transmembrane domains and two nucleotide-binding domains. The single glycosylation site is represented as Ψ in the extracellular N-terminus. The insertion site for the biotin ligase acceptor peptide is also shown in the N-terminus at proline 4. B, a sequence alignment of known PDZ-containing ABCC family members is shown. The Type I consensus is shown above the alignment as is represented as: A, acidic; P, polar, X, any; and H, hydrophobic amino acids. The PXE-associate G1501S site is highlighted in red. C, representative western blots of the wildtype and Δ6-COOH ABCC6 proteins are shown after expression in HEK293 cells. The core and complexly glycosylated species are indicated on the left by B and C, respectively. Two exposures are shown for the Δ6-COOH to illustrate the formation of both band B and C at low levels in the mutant protein. D, endoglycosidase assays confirm the glycosylation state and differential electrophoretic migration of the ABCC6 proteins. The differential digestion of the band C protein by EndoH and PNGaseF demonstrates complex glycosylation, consistent with trafficking through the Golgi. The N15D substitution blocks N-linked glycosylation and is a reference for the unglycosylated wildtype and Δ6-COOH proteins. E , representative western blots from cell surface biotinylation experiments are shown. Cell surface expression is shown for cells mock transfected (CNTL) or transfected with wildtype or Δ6-COOH ABCC6, Cell Surface . Whole cell lysates are shown, Total , from samples prior to streptavidin pull-down. The control samples are taken from non-adjacent wells on a single gel/film. F, immunofluorescence images of the wildtype and Δ6-COOH proteins are shown. The ABCC6 proteins are shown in green, phalloidin is shown in red, and DAPI is shown in blue. Colocalization of the ABCC6 protein with phalloidin is consistent with ABCC6 trafficking to the cell surface in the wildtype protein and is decreased by the Δ6-COOH mutant. G, immunofluorescence images of the wildtype and Δ6-COOH ABCC6 proteins are shown after expression in polarized MDCK cells. The wildtype ABCC6 protein localizes to the basolateral membrane in polarized MDCK cells, left. The Δ6-COOH protein shows significant intracellular staining and a loss of basolateral targeting in MDCK cells, right . Both X–Y, top , and X–Z, bottom , images are shown. For G , ABCC6 is stained in green and ZO1 is shown in red . Western blots are representative of samples from at least three independent experiments.
    Figure Legend Snippet: Alteration of ABCC6 trafficking by the PDZ-like C-terminus. To evaluate the potential role of the PDZ-like sequences at the C-terminus of ABCC6, wildtype and mutant proteins were expressed in HEK293 cells and evaluated by western blotting and immunofluorescence. A, a cartoon illustrating the domain organization and topology of ABCC6 is shown. ABCC6 contains three transmembrane domains and two nucleotide-binding domains. The single glycosylation site is represented as Ψ in the extracellular N-terminus. The insertion site for the biotin ligase acceptor peptide is also shown in the N-terminus at proline 4. B, a sequence alignment of known PDZ-containing ABCC family members is shown. The Type I consensus is shown above the alignment as is represented as: A, acidic; P, polar, X, any; and H, hydrophobic amino acids. The PXE-associate G1501S site is highlighted in red. C, representative western blots of the wildtype and Δ6-COOH ABCC6 proteins are shown after expression in HEK293 cells. The core and complexly glycosylated species are indicated on the left by B and C, respectively. Two exposures are shown for the Δ6-COOH to illustrate the formation of both band B and C at low levels in the mutant protein. D, endoglycosidase assays confirm the glycosylation state and differential electrophoretic migration of the ABCC6 proteins. The differential digestion of the band C protein by EndoH and PNGaseF demonstrates complex glycosylation, consistent with trafficking through the Golgi. The N15D substitution blocks N-linked glycosylation and is a reference for the unglycosylated wildtype and Δ6-COOH proteins. E , representative western blots from cell surface biotinylation experiments are shown. Cell surface expression is shown for cells mock transfected (CNTL) or transfected with wildtype or Δ6-COOH ABCC6, Cell Surface . Whole cell lysates are shown, Total , from samples prior to streptavidin pull-down. The control samples are taken from non-adjacent wells on a single gel/film. F, immunofluorescence images of the wildtype and Δ6-COOH proteins are shown. The ABCC6 proteins are shown in green, phalloidin is shown in red, and DAPI is shown in blue. Colocalization of the ABCC6 protein with phalloidin is consistent with ABCC6 trafficking to the cell surface in the wildtype protein and is decreased by the Δ6-COOH mutant. G, immunofluorescence images of the wildtype and Δ6-COOH ABCC6 proteins are shown after expression in polarized MDCK cells. The wildtype ABCC6 protein localizes to the basolateral membrane in polarized MDCK cells, left. The Δ6-COOH protein shows significant intracellular staining and a loss of basolateral targeting in MDCK cells, right . Both X–Y, top , and X–Z, bottom , images are shown. For G , ABCC6 is stained in green and ZO1 is shown in red . Western blots are representative of samples from at least three independent experiments.

    Techniques Used: Mutagenesis, Western Blot, Immunofluorescence, Binding Assay, Sequencing, Expressing, Migration, Transfection, Staining

    Impact of the C-terminus on ABCC6 degradation. ABCC6 degradation was evaluated after treatment with a proteasome inhibitor (lactacystin) or a combination of lysosomal protease inhibitors, (leupeptin and pepstatin) and assessed by western blotting. A,B, inhibition of the proteasome by lactacystin results in an accumulation of the ER-resident, band B protein in the mutant ABCC6. A, increasing lactacystin concentrations from 0–10 µM, results in an accumulation of the band B form of the mutant ABCC6 protein as seen by western blotting. No increase in the formation of the band C, complexly glycosylated protein is seen for either wildtype or mutant ABCC6 with lactacystin treatment. B, immunofluorescence of the ABCC6 proteins reveals the wildtype and mutant accumulate after proteasome inhibition, but the mutant fails to redistribute to the cell surface. ABCC6 is shown in green, phalloidin is shown in red and DAPI is shown in blue. C,D, lysosomal inhibition results in an increase in the complexly glycosylated, band C protein for both wildtype and mutant ABCC6. C, a dose response of leupeptin/pepstatin treatment is shown from 0–100 µM leupeptin treatment in the presence of 1 µg/ml pepstatin. Increasing pepstatin concentrations resulted in an increase in the band C ABCC6 protein. D, immunofluorescence of HEK293 cells treated with leupeptin/pepstatin is shown. Treatment with leupeptin/pepstatin resulted in an increase in the quantities of ABCC6 intracellularly. ABCC6 is shown in green, phalloidin is shown in red and DAPI is shown in blue.
    Figure Legend Snippet: Impact of the C-terminus on ABCC6 degradation. ABCC6 degradation was evaluated after treatment with a proteasome inhibitor (lactacystin) or a combination of lysosomal protease inhibitors, (leupeptin and pepstatin) and assessed by western blotting. A,B, inhibition of the proteasome by lactacystin results in an accumulation of the ER-resident, band B protein in the mutant ABCC6. A, increasing lactacystin concentrations from 0–10 µM, results in an accumulation of the band B form of the mutant ABCC6 protein as seen by western blotting. No increase in the formation of the band C, complexly glycosylated protein is seen for either wildtype or mutant ABCC6 with lactacystin treatment. B, immunofluorescence of the ABCC6 proteins reveals the wildtype and mutant accumulate after proteasome inhibition, but the mutant fails to redistribute to the cell surface. ABCC6 is shown in green, phalloidin is shown in red and DAPI is shown in blue. C,D, lysosomal inhibition results in an increase in the complexly glycosylated, band C protein for both wildtype and mutant ABCC6. C, a dose response of leupeptin/pepstatin treatment is shown from 0–100 µM leupeptin treatment in the presence of 1 µg/ml pepstatin. Increasing pepstatin concentrations resulted in an increase in the band C ABCC6 protein. D, immunofluorescence of HEK293 cells treated with leupeptin/pepstatin is shown. Treatment with leupeptin/pepstatin resulted in an increase in the quantities of ABCC6 intracellularly. ABCC6 is shown in green, phalloidin is shown in red and DAPI is shown in blue.

    Techniques Used: Western Blot, Inhibition, Mutagenesis, Immunofluorescence

    Rescue of ABCC6 trafficking by low temperature expression. Low temperature expression was used to further evaluate ABCC6 trafficking in HEK293 cells. A, western blots showing the expression of the wildtype and Δ6-COOH ABCC6 proteins at 37°C and 27°C. Expression of the wildtype protein at 27°C results in an increase in relative quantities of the core glycosylated, band B protein. Expressing the mutant protein at low temperature resulted in an increase in complexly glycosylated protein (band C), consistent with an increase in the formation or stabilization of this protein. B, indirect immunofluorescence of ABCC6 confirming the trafficking of the wildtype and mutant proteins is shown. Consistent with the western blotting, expression at low temperature results in redistribution of the mutant protein towards the plasma membrane. ABCC6 is shown in green, phalloidin is shown in red and DAPI is shown in blue.
    Figure Legend Snippet: Rescue of ABCC6 trafficking by low temperature expression. Low temperature expression was used to further evaluate ABCC6 trafficking in HEK293 cells. A, western blots showing the expression of the wildtype and Δ6-COOH ABCC6 proteins at 37°C and 27°C. Expression of the wildtype protein at 27°C results in an increase in relative quantities of the core glycosylated, band B protein. Expressing the mutant protein at low temperature resulted in an increase in complexly glycosylated protein (band C), consistent with an increase in the formation or stabilization of this protein. B, indirect immunofluorescence of ABCC6 confirming the trafficking of the wildtype and mutant proteins is shown. Consistent with the western blotting, expression at low temperature results in redistribution of the mutant protein towards the plasma membrane. ABCC6 is shown in green, phalloidin is shown in red and DAPI is shown in blue.

    Techniques Used: Expressing, Western Blot, Mutagenesis, Immunofluorescence

    Regulation of cell surface stability by the C-terminus. Cell surface stability was evaluated by selective biotinylation of the ABCC6 protein using the BirA ligase and acceptor peptide in the extracellular N-terminus (see Figure 1A ). A, western blots of the wildtype and Δ6-COOH ABCC6 protein with the BLAP tag are shown. The inclusion of the BLAP tag in the N-terminus had no detectable effect on the trafficking of the wildtype or mutant ABCC6 proteins evaluated by western blotting. B, fluorophore-conjugated streptavidin was applied to the culture media and cell surface expression of the BLAP ABCC6 protein was evaluated by fluorescence microscopy. Consistent with western blotting, no detectable differences were seen between the BLAP tagged and untagged ABCC6 proteins. The wildtype protein expressed robustly in HEK 293 cells, while the mutant protein was only labeled in a small fraction of cells transfected. C, fluorescence analysis of the timecourse of ABCC6 internalization and degradation from the cell surface is shown. The BLAP tagged proteins were sequentially labeled with fluorophore-conjugated streptavidin. Initial staining, time zero, was performed using AlexaFluor-488, green , and secondary labeling was performed using AlexaFluor-555, red. The internalization and degradation of ABCC6 could be seen over the course of 4–18 hours as the loss of green signal. D, western blots of cell surface labeled ABCC6 are shown. Streptavidin was incubated extracellularly on intact HEK293 and the BLAP-tagged ABCC6 protein was bound and washed. The lystes were subjected to SDS-PAGE and western blotting. The conjugated ABCC6-streptavidin complex could be distinguished readily from the total ABCC6 protein, allowing for the evaluation of plasma-membrane ABCC6 protein. Proteins were labeled, washed and incubated for zero to eight hours before lysis. Negative controls included expression of the BLAP-ABCC6 protein without streptavidin treatment (C1) and mock-transfected HEK293 cells treated with BirA and streptavidin (C2). Both negative controls showed no staining, consistent with specific detection of labeled BLAP-tagged ABCC6 in the experimental samples.
    Figure Legend Snippet: Regulation of cell surface stability by the C-terminus. Cell surface stability was evaluated by selective biotinylation of the ABCC6 protein using the BirA ligase and acceptor peptide in the extracellular N-terminus (see Figure 1A ). A, western blots of the wildtype and Δ6-COOH ABCC6 protein with the BLAP tag are shown. The inclusion of the BLAP tag in the N-terminus had no detectable effect on the trafficking of the wildtype or mutant ABCC6 proteins evaluated by western blotting. B, fluorophore-conjugated streptavidin was applied to the culture media and cell surface expression of the BLAP ABCC6 protein was evaluated by fluorescence microscopy. Consistent with western blotting, no detectable differences were seen between the BLAP tagged and untagged ABCC6 proteins. The wildtype protein expressed robustly in HEK 293 cells, while the mutant protein was only labeled in a small fraction of cells transfected. C, fluorescence analysis of the timecourse of ABCC6 internalization and degradation from the cell surface is shown. The BLAP tagged proteins were sequentially labeled with fluorophore-conjugated streptavidin. Initial staining, time zero, was performed using AlexaFluor-488, green , and secondary labeling was performed using AlexaFluor-555, red. The internalization and degradation of ABCC6 could be seen over the course of 4–18 hours as the loss of green signal. D, western blots of cell surface labeled ABCC6 are shown. Streptavidin was incubated extracellularly on intact HEK293 and the BLAP-tagged ABCC6 protein was bound and washed. The lystes were subjected to SDS-PAGE and western blotting. The conjugated ABCC6-streptavidin complex could be distinguished readily from the total ABCC6 protein, allowing for the evaluation of plasma-membrane ABCC6 protein. Proteins were labeled, washed and incubated for zero to eight hours before lysis. Negative controls included expression of the BLAP-ABCC6 protein without streptavidin treatment (C1) and mock-transfected HEK293 cells treated with BirA and streptavidin (C2). Both negative controls showed no staining, consistent with specific detection of labeled BLAP-tagged ABCC6 in the experimental samples.

    Techniques Used: Western Blot, Mutagenesis, Expressing, Fluorescence, Microscopy, Labeling, Transfection, Staining, Incubation, SDS Page, Lysis

    Impact of the C-terminus on ABCC6 turnover. Protein turnover was evaluated by cyclohexamide chase experiments after expression in HEK293 cells. A, western blots of the wildtype and Δ6-COOH proteins are shown after 0, 4, 8, and 18 hours of cyclohexamide treatment. The wildtype protein shows minimal changes after 18 hours of cyclohexamide treatment, while the mutant is decreased by ∼80% over this timecourse. The loss of band B protein in the mutant is consistent with the inhibition of new ABCC6 synthesis over the timecourse of treatment resulting from cyclohexamide treatment. B, summary data for cyclohexamide chase experiments are shown. Between 8 and 18 hours of cyclohexamide chase, the Δ6-COOH mutant protein is diminished by ∼80% relative to the wildtype protein. Data shown are mean +/− standard deviation from n = 3 experiments.
    Figure Legend Snippet: Impact of the C-terminus on ABCC6 turnover. Protein turnover was evaluated by cyclohexamide chase experiments after expression in HEK293 cells. A, western blots of the wildtype and Δ6-COOH proteins are shown after 0, 4, 8, and 18 hours of cyclohexamide treatment. The wildtype protein shows minimal changes after 18 hours of cyclohexamide treatment, while the mutant is decreased by ∼80% over this timecourse. The loss of band B protein in the mutant is consistent with the inhibition of new ABCC6 synthesis over the timecourse of treatment resulting from cyclohexamide treatment. B, summary data for cyclohexamide chase experiments are shown. Between 8 and 18 hours of cyclohexamide chase, the Δ6-COOH mutant protein is diminished by ∼80% relative to the wildtype protein. Data shown are mean +/− standard deviation from n = 3 experiments.

    Techniques Used: Expressing, Western Blot, Mutagenesis, Inhibition, Standard Deviation

    9) Product Images from "Regulation of CDKN2B expression by interaction of Arnt with Miz-1 - a basis for functional integration between the HIF and Myc gene regulatory pathways"

    Article Title: Regulation of CDKN2B expression by interaction of Arnt with Miz-1 - a basis for functional integration between the HIF and Myc gene regulatory pathways

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-13-54

    Arnt and Miz-1 interact. A) Schematic representation of motifs within native and mutant constructs of Arnt. The human Arnt used in this study consists of 774 amino acids and the location of its helix-loop-helix (bHLH), Per-Arnt-Sim (PAS) and the glutamine rich (Q-rich) domains are shown. The amino acid mutations in “2xmut” and “4xmut” are indicated in bold in the Helix II sequence. B) Miz-1/GFP and wt or mutated variants of Arnt/Flag were overexpressed as indicated. Immunoprecipitation (IP) was performed using anti-GFP and immunoblotting (IB) was performed with anti-Flag (upper panel). Input (4%) was run on a parallel gel and IB was performed with anti-Flag (middle panel). In order to verify Miz-1/GFP IP in the different samples, the co-IP membrane was stripped and incubated with anti-GFP (lower panel). C) HEK293 cells were co-transfected with Miz-1/Flag and Arnt/CFP. Cells were subjected to confocal microscopy to visualize Texas-red fluorescence (for Miz-1/Flag; left panel) or cyan fluorescence (for Arnt/CFP; middle panel). The right panel shows the merged image. The borders of the cytoplasm of the cells are marked in white, while the borders of nuclei are marked in yellow.
    Figure Legend Snippet: Arnt and Miz-1 interact. A) Schematic representation of motifs within native and mutant constructs of Arnt. The human Arnt used in this study consists of 774 amino acids and the location of its helix-loop-helix (bHLH), Per-Arnt-Sim (PAS) and the glutamine rich (Q-rich) domains are shown. The amino acid mutations in “2xmut” and “4xmut” are indicated in bold in the Helix II sequence. B) Miz-1/GFP and wt or mutated variants of Arnt/Flag were overexpressed as indicated. Immunoprecipitation (IP) was performed using anti-GFP and immunoblotting (IB) was performed with anti-Flag (upper panel). Input (4%) was run on a parallel gel and IB was performed with anti-Flag (middle panel). In order to verify Miz-1/GFP IP in the different samples, the co-IP membrane was stripped and incubated with anti-GFP (lower panel). C) HEK293 cells were co-transfected with Miz-1/Flag and Arnt/CFP. Cells were subjected to confocal microscopy to visualize Texas-red fluorescence (for Miz-1/Flag; left panel) or cyan fluorescence (for Arnt/CFP; middle panel). The right panel shows the merged image. The borders of the cytoplasm of the cells are marked in white, while the borders of nuclei are marked in yellow.

    Techniques Used: Mutagenesis, Construct, Sequencing, Immunoprecipitation, Co-Immunoprecipitation Assay, Incubation, Transfection, Confocal Microscopy, Fluorescence

    10) Product Images from "d-Amino acid oxidase and serine racemase in human brain: normal distribution and altered expression in schizophrenia"

    Article Title: d-Amino acid oxidase and serine racemase in human brain: normal distribution and altered expression in schizophrenia

    Journal: The European Journal of Neuroscience

    doi: 10.1111/j.1460-9568.2007.05769.x

    Detection of DAO and SRR. (A) Validation of DAO antibody using recombinant flag-DAO expressed in HEK293 cells. Detection with anti-DAO (lanes 1 and 2) and anti-flag (lanes 3 and 4). Flag-DAO is detected as a ∼42-kDa band with anti-DAO (lane 1) and anti-flag (lane 3) in flag-DAO transfected HEK293 cells but not mock transfected cells (lanes 2 and 4). Expressed untagged DAO is detected as a ∼39-kDa band, of predicted size for DAO, with anti-DAO antibody (lane 1 lower band) but not anti-flag (lane 3). (B) Validation of DAO antibody using human tissue samples (Clontech protein medleys); lane 1, cerebellum; lane 2, amygdala; lane 3, hippocampus; lane 4, thalamus; lane 5, spinal cord; lane 6, lung; lane 7, heart; lane 8, kidney. A specific signal for DAO was detected at ∼39 kDa in the cerebellum, spinal cord and kidney. Blots were re-probed with anti-β-actin as a loading control (lower panel). (C) Representative Western blots of DAO (lanes 1 and 2), DAO pre-adsorption control (lanes 3 and 4), and SRR (lanes 5–6) in protein extracts of post-mortem cerebellar tissue from human (lanes 1, 3 and 5) and rat (lanes 2, 4 and 6). DAO was detected at ∼39 kDa and SRR at ∼38 kDa. Blots were simultaneously probed for cyclophilin as a loading control (lower band), which was detected at ∼20 kDa.
    Figure Legend Snippet: Detection of DAO and SRR. (A) Validation of DAO antibody using recombinant flag-DAO expressed in HEK293 cells. Detection with anti-DAO (lanes 1 and 2) and anti-flag (lanes 3 and 4). Flag-DAO is detected as a ∼42-kDa band with anti-DAO (lane 1) and anti-flag (lane 3) in flag-DAO transfected HEK293 cells but not mock transfected cells (lanes 2 and 4). Expressed untagged DAO is detected as a ∼39-kDa band, of predicted size for DAO, with anti-DAO antibody (lane 1 lower band) but not anti-flag (lane 3). (B) Validation of DAO antibody using human tissue samples (Clontech protein medleys); lane 1, cerebellum; lane 2, amygdala; lane 3, hippocampus; lane 4, thalamus; lane 5, spinal cord; lane 6, lung; lane 7, heart; lane 8, kidney. A specific signal for DAO was detected at ∼39 kDa in the cerebellum, spinal cord and kidney. Blots were re-probed with anti-β-actin as a loading control (lower panel). (C) Representative Western blots of DAO (lanes 1 and 2), DAO pre-adsorption control (lanes 3 and 4), and SRR (lanes 5–6) in protein extracts of post-mortem cerebellar tissue from human (lanes 1, 3 and 5) and rat (lanes 2, 4 and 6). DAO was detected at ∼39 kDa and SRR at ∼38 kDa. Blots were simultaneously probed for cyclophilin as a loading control (lower band), which was detected at ∼20 kDa.

    Techniques Used: Recombinant, Transfection, Western Blot, Adsorption

    11) Product Images from "Deubiquitination of CXCR4 by USP14 Is Critical for Both CXCL12-induced CXCR4 Degradation and Chemotaxis but Not ERK Activation *"

    Article Title: Deubiquitination of CXCR4 by USP14 Is Critical for Both CXCL12-induced CXCR4 Degradation and Chemotaxis but Not ERK Activation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M808507200

    CXCL12 enhances apparent USP14 co-localization with CXCR4. HEK293 cells stably expressing EGFP-CXCR4 and transiently transfected with HA-USP14 were treated without (control) or with CXCL12 (10 n m ) for the indicated time intervals. The cells were fixed and evaluated using immunohistochemistry via confocal microscopy as detailed under “Experimental Procedures.” Representative laser-scanning confocal micrographs demonstrating the distribution of EGFP-CXCR4 ( green ), USP14 ( red ), and overlay ( yellow ) are shown. Images were processed using Photoshop software.
    Figure Legend Snippet: CXCL12 enhances apparent USP14 co-localization with CXCR4. HEK293 cells stably expressing EGFP-CXCR4 and transiently transfected with HA-USP14 were treated without (control) or with CXCL12 (10 n m ) for the indicated time intervals. The cells were fixed and evaluated using immunohistochemistry via confocal microscopy as detailed under “Experimental Procedures.” Representative laser-scanning confocal micrographs demonstrating the distribution of EGFP-CXCR4 ( green ), USP14 ( red ), and overlay ( yellow ) are shown. Images were processed using Photoshop software.

    Techniques Used: Stable Transfection, Expressing, Transfection, Immunohistochemistry, Confocal Microscopy, Software

    USP14 modulates CXCR4 ubiquitination. A, antibody directed against endogenous ubiquitin reveals the time-dependent ubiquitination of Myc-CXCR4 ( upper panel ) in response to CXCL12 (10 n m ) treatment of HEK293 cells (see “Experimental Procedures”). The lower panel , obtained by reprobing the Western blot with an anti-Myc antibody directed against the Myc-CXCR4, readily detects the receptor protein migrating at ∼45,000, but not the higher molecular mass “ladders” at 54 kDa and above. IP , immunoprecipitation; IB , immunoblot. B, quantitation of the relative density of bands representing CXCR4-endogenous Ub complexes was determined by densitometric scanning (•; see “Experimental Procedures”). Superimposed on these data are the findings from the time course of USP14 association with the CXCR4 in CXCL12-exposed cells (○), reported in Fig. 1 B , for comparison. C, overexpression of USP14 eliminates detectable CXCR4 ubiquitination in response to CXCL12. HEK293 cells stably expressing Myc-CXCR4 and transiently transfected with vector alone ( Vector ) or HA-USP14 were incubated for 10 min with CXCL12 as in A . This incubation was terminated immediately for some samples ( 10 min, no recovery ) but allowed to continue after washing away the CXCL12, for 60 min ( 10 min, + 60 min recovery ). The gel data shown below the bar graph , from one representative experiment, confirm that transfection of the HEK293 cells with the cDNA encoding HA-USP14 indeed leads to overexpression of this enzyme. D, RNA interference knockdown of endogenous USP14 eliminates deubiquitination of CXCL12-evoked ubiquitination of Myc-CXCR4. HEK293 cells transfected with scrambled ( control ) siRNA or USP14 siRNAs were treated without CXCL12 ( none ) or with 10 n m CXCL12 for 10 min ( 10 min, no recovery ) or 10 min followed by a 60-min recovery period ( 10 min, + 60 min recovery ) as described under “Experimental Procedures.” The panel below the bar graph provides a representative gel that confirms the ability of the siRNA construct to successfully reduce the expression of the USP14 protein in these cells under these conditions. Myc-CXCR4 was isolated by immunoprecipitation with an anti-Myc antibody, and the CXCR4-Ub complexes were quantified by Western blot using an antibody against endogenous ubiquitin. Blots were stripped and reprobed using a Myc antibody to evaluate loading of Myc-CXCR4. Data in B-D represent the mean ± S.E. from three independent experiments. * , p
    Figure Legend Snippet: USP14 modulates CXCR4 ubiquitination. A, antibody directed against endogenous ubiquitin reveals the time-dependent ubiquitination of Myc-CXCR4 ( upper panel ) in response to CXCL12 (10 n m ) treatment of HEK293 cells (see “Experimental Procedures”). The lower panel , obtained by reprobing the Western blot with an anti-Myc antibody directed against the Myc-CXCR4, readily detects the receptor protein migrating at ∼45,000, but not the higher molecular mass “ladders” at 54 kDa and above. IP , immunoprecipitation; IB , immunoblot. B, quantitation of the relative density of bands representing CXCR4-endogenous Ub complexes was determined by densitometric scanning (•; see “Experimental Procedures”). Superimposed on these data are the findings from the time course of USP14 association with the CXCR4 in CXCL12-exposed cells (○), reported in Fig. 1 B , for comparison. C, overexpression of USP14 eliminates detectable CXCR4 ubiquitination in response to CXCL12. HEK293 cells stably expressing Myc-CXCR4 and transiently transfected with vector alone ( Vector ) or HA-USP14 were incubated for 10 min with CXCL12 as in A . This incubation was terminated immediately for some samples ( 10 min, no recovery ) but allowed to continue after washing away the CXCL12, for 60 min ( 10 min, + 60 min recovery ). The gel data shown below the bar graph , from one representative experiment, confirm that transfection of the HEK293 cells with the cDNA encoding HA-USP14 indeed leads to overexpression of this enzyme. D, RNA interference knockdown of endogenous USP14 eliminates deubiquitination of CXCL12-evoked ubiquitination of Myc-CXCR4. HEK293 cells transfected with scrambled ( control ) siRNA or USP14 siRNAs were treated without CXCL12 ( none ) or with 10 n m CXCL12 for 10 min ( 10 min, no recovery ) or 10 min followed by a 60-min recovery period ( 10 min, + 60 min recovery ) as described under “Experimental Procedures.” The panel below the bar graph provides a representative gel that confirms the ability of the siRNA construct to successfully reduce the expression of the USP14 protein in these cells under these conditions. Myc-CXCR4 was isolated by immunoprecipitation with an anti-Myc antibody, and the CXCR4-Ub complexes were quantified by Western blot using an antibody against endogenous ubiquitin. Blots were stripped and reprobed using a Myc antibody to evaluate loading of Myc-CXCR4. Data in B-D represent the mean ± S.E. from three independent experiments. * , p

    Techniques Used: Western Blot, Immunoprecipitation, Quantitation Assay, Over Expression, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Incubation, Construct, Isolation

    ERK activation by CXCR4 occurs independently of the ability of the CXCR4 to be ubiquitinated. A, ERK activation was evaluated in HEK293 cells transiently transfected with HA-WT CXCR4 or HA-3K/R CXCR4 and stimulated with CXCL12 (10 n m ) for 0, 5, 15, 30, or 60 min, and ERK activity in cell lysates assessed was as described under “Experimental Procedures.” B , quantitation of ERK activation was based on the amount of ERK detected using an anti-P-ERK antibody; total ERK, assessed using the ERK2 antibody, was indistinguishable in all conditions, and thus the data were not normalized to total ERK. Data are mean ± S.E. from three independent experiments.
    Figure Legend Snippet: ERK activation by CXCR4 occurs independently of the ability of the CXCR4 to be ubiquitinated. A, ERK activation was evaluated in HEK293 cells transiently transfected with HA-WT CXCR4 or HA-3K/R CXCR4 and stimulated with CXCL12 (10 n m ) for 0, 5, 15, 30, or 60 min, and ERK activity in cell lysates assessed was as described under “Experimental Procedures.” B , quantitation of ERK activation was based on the amount of ERK detected using an anti-P-ERK antibody; total ERK, assessed using the ERK2 antibody, was indistinguishable in all conditions, and thus the data were not normalized to total ERK. Data are mean ± S.E. from three independent experiments.

    Techniques Used: Activation Assay, Transfection, Activity Assay, Quantitation Assay

    USP14 prevents CXCL12-mediated EGFP-CXCR4 degradation and increases the steady state level of the receptor. A, HEK293 cells stably expressing EGFP-CXCR4 were treated with CXCL12 for 8 h (+); this prolonged incubation allowed detection of CXCL12-evoked receptor down-regulation. EGFP-CXCR4 levels were detected by Western blot using an anti-EGFP antibody. B, quantitation of the relative amount of CXCR4 was determined by densitometric scanning as outlined under “Experimental Procedures.” Data are mean ± S.E. from three independent experiments. * , p
    Figure Legend Snippet: USP14 prevents CXCL12-mediated EGFP-CXCR4 degradation and increases the steady state level of the receptor. A, HEK293 cells stably expressing EGFP-CXCR4 were treated with CXCL12 for 8 h (+); this prolonged incubation allowed detection of CXCL12-evoked receptor down-regulation. EGFP-CXCR4 levels were detected by Western blot using an anti-EGFP antibody. B, quantitation of the relative amount of CXCR4 was determined by densitometric scanning as outlined under “Experimental Procedures.” Data are mean ± S.E. from three independent experiments. * , p

    Techniques Used: Stable Transfection, Expressing, Incubation, Western Blot, Quantitation Assay

    CXCR4 selectively interacts with USP14 via the C terminus of the receptor. HEK293 cells stably expressing Myc-CXCR4 were exposed to CXCL12 (10 n m ) for the indicated time intervals ( A-C ). Myc-CXCR4 was immunoprecipitated ( IP ) from cell lysates using a mouse anti-Myc antibody (see “Experimental Procedures”). A, CXCL12 causes a time-dependent association of CXCR14 with USP14. The amount of co-precipitated USP14 protein was detected by Western blotting for the HA epitope on USP14. The membrane was stripped and reprobed using a rabbit anti-Myc antibody to evaluate Myc-CXCR4 loading. The migration of molecular weight markers is shown to the left of the gel. Data shown are representative of one experiment performed six times. IB , immunoblot. B, quantitation of the relative amount of USP14 co-precipitated with CXCR4 was determined by densitometric scanning as outlined under “Experimental Procedures”; n = 6. C, selectivity of USP-isoform interaction with Myc-CXCR4. Experiments were performed as in A and described in detail under “Experimental Procedures.” There was no detectable interaction of the CXCR4 with USP7 (data not shown). Co-precipitated HA-USP2a (▴, n = 4) and His-USP4 (▪, n = 3) were detected using anti-HA and anti-His antibodies (see “Experimental Procedures”). D , USP14 interacts with the C terminus of CXCR4; GSH-Sepharose-bound GST ( lane 2 , control) or GST-CXCR4 C-terminal fusion protein ( lane 3 ) was incubated with HEK293 cell lysates prepared from control ( i.e. not stimulated by CXCL12 ligand), as described under “Experimental Procedures.” Upper panel, HA-USP14 was detected by Western blotting using an anti-HA antibody. Lower panel, GST was detected using a rabbit anti-GST antibody. An aliquot of the cell lysate is shown in lane 1 . Data shown in B and C are mean ± S.E. from the number of independent experiments outlined above. * , p
    Figure Legend Snippet: CXCR4 selectively interacts with USP14 via the C terminus of the receptor. HEK293 cells stably expressing Myc-CXCR4 were exposed to CXCL12 (10 n m ) for the indicated time intervals ( A-C ). Myc-CXCR4 was immunoprecipitated ( IP ) from cell lysates using a mouse anti-Myc antibody (see “Experimental Procedures”). A, CXCL12 causes a time-dependent association of CXCR14 with USP14. The amount of co-precipitated USP14 protein was detected by Western blotting for the HA epitope on USP14. The membrane was stripped and reprobed using a rabbit anti-Myc antibody to evaluate Myc-CXCR4 loading. The migration of molecular weight markers is shown to the left of the gel. Data shown are representative of one experiment performed six times. IB , immunoblot. B, quantitation of the relative amount of USP14 co-precipitated with CXCR4 was determined by densitometric scanning as outlined under “Experimental Procedures”; n = 6. C, selectivity of USP-isoform interaction with Myc-CXCR4. Experiments were performed as in A and described in detail under “Experimental Procedures.” There was no detectable interaction of the CXCR4 with USP7 (data not shown). Co-precipitated HA-USP2a (▴, n = 4) and His-USP4 (▪, n = 3) were detected using anti-HA and anti-His antibodies (see “Experimental Procedures”). D , USP14 interacts with the C terminus of CXCR4; GSH-Sepharose-bound GST ( lane 2 , control) or GST-CXCR4 C-terminal fusion protein ( lane 3 ) was incubated with HEK293 cell lysates prepared from control ( i.e. not stimulated by CXCL12 ligand), as described under “Experimental Procedures.” Upper panel, HA-USP14 was detected by Western blotting using an anti-HA antibody. Lower panel, GST was detected using a rabbit anti-GST antibody. An aliquot of the cell lysate is shown in lane 1 . Data shown in B and C are mean ± S.E. from the number of independent experiments outlined above. * , p

    Techniques Used: Stable Transfection, Expressing, Immunoprecipitation, Western Blot, Migration, Molecular Weight, Quantitation Assay, Incubation

    CXCR4-Ub cycle is essential for CXCR4-mediated chemotaxis. A, chemotaxis was evaluated in HEK293 cells stably expressing Myc-CXCR4 (and endogenous levels of USP14, i.e. “vector alone”) or in cells overexpressing HA-USP14 as described in detail under “Experimental Procedures.” Overexpression of USP14, which dramatically reduces CXCL12-evoked CXCR4 ubiquitination ( cf. Fig. 3 C ), also dramatically attenuates CXCL12-evoked chemotaxis. B, knockdown of endogenous USP14 expression with USP14-directed siRNA, which leads to enhanced CXCL12-induced CXCR4 ubiquitination ( cf. Fig. 3 D ), also significantly reduces CXCL12-induced chemotaxis. C, chemotaxis in response to CXCL12 was evaluated in cells expressing an HA-WT CXCR4 and in cells expressing a 3K/R mutant receptor, as described under “Experimental Procedures.” Taken together, A and B suggest that the CXCR4-Ub cycle, and not a particular ubiquitinated state of CXCR4, is essential for CXCL12-mediated chemotaxis, and the data in C confirm that it is the CXCR4 molecule itself that must undergo a ubiquitination/deubiquitination cycle for chemotaxis to occur. Values represent the mean ± S.E. from three independent experiments performed in duplicate. All chemotaxis data are expressed as the chemotactic index, which is calculated as the ratio of the number of cells that migrate across the Boyden chamber in the presence of CXCL12 at a given concentration compared with the number of cells migrating in the absence of CXCL12; the value of 1 means that there was no migration greater than that observed in control, nonstimulated cells. Data were analyzed using Student's unpaired t test. * , p
    Figure Legend Snippet: CXCR4-Ub cycle is essential for CXCR4-mediated chemotaxis. A, chemotaxis was evaluated in HEK293 cells stably expressing Myc-CXCR4 (and endogenous levels of USP14, i.e. “vector alone”) or in cells overexpressing HA-USP14 as described in detail under “Experimental Procedures.” Overexpression of USP14, which dramatically reduces CXCL12-evoked CXCR4 ubiquitination ( cf. Fig. 3 C ), also dramatically attenuates CXCL12-evoked chemotaxis. B, knockdown of endogenous USP14 expression with USP14-directed siRNA, which leads to enhanced CXCL12-induced CXCR4 ubiquitination ( cf. Fig. 3 D ), also significantly reduces CXCL12-induced chemotaxis. C, chemotaxis in response to CXCL12 was evaluated in cells expressing an HA-WT CXCR4 and in cells expressing a 3K/R mutant receptor, as described under “Experimental Procedures.” Taken together, A and B suggest that the CXCR4-Ub cycle, and not a particular ubiquitinated state of CXCR4, is essential for CXCL12-mediated chemotaxis, and the data in C confirm that it is the CXCR4 molecule itself that must undergo a ubiquitination/deubiquitination cycle for chemotaxis to occur. Values represent the mean ± S.E. from three independent experiments performed in duplicate. All chemotaxis data are expressed as the chemotactic index, which is calculated as the ratio of the number of cells that migrate across the Boyden chamber in the presence of CXCL12 at a given concentration compared with the number of cells migrating in the absence of CXCL12; the value of 1 means that there was no migration greater than that observed in control, nonstimulated cells. Data were analyzed using Student's unpaired t test. * , p

    Techniques Used: Chemotaxis Assay, Stable Transfection, Expressing, Over Expression, Mutagenesis, Concentration Assay, Migration

    12) Product Images from "Subtype-specific regulation of P2X3 and P2X2/3 receptors by phosphoinositides in peripheral nociceptors"

    Article Title: Subtype-specific regulation of P2X3 and P2X2/3 receptors by phosphoinositides in peripheral nociceptors

    Journal: Molecular Pain

    doi: 10.1186/1744-8069-5-47

    Sensitivity of P2X3 current to PIP 2 depletion in HEK293 cells is reversed by R356Q mutation . A) Pooled data of wild-type and mutant P2X3 responses to 10 μM ATP expressed in HEK293 cells (N = 5–11). B) Representative surface biotinylation data of wild-type and mutant P2X3 receptor expression. C) Sample traces showing 35 μM wortmannin-induced rundown of wild-type P2X3 currents in HEK293 cells. D) Quantitative results. Rundown of wild-type and R356Q mutant P2X3 current in response to 35 μM wortmannin incubation (N = 5–6). (**, P
    Figure Legend Snippet: Sensitivity of P2X3 current to PIP 2 depletion in HEK293 cells is reversed by R356Q mutation . A) Pooled data of wild-type and mutant P2X3 responses to 10 μM ATP expressed in HEK293 cells (N = 5–11). B) Representative surface biotinylation data of wild-type and mutant P2X3 receptor expression. C) Sample traces showing 35 μM wortmannin-induced rundown of wild-type P2X3 currents in HEK293 cells. D) Quantitative results. Rundown of wild-type and R356Q mutant P2X3 current in response to 35 μM wortmannin incubation (N = 5–6). (**, P

    Techniques Used: Mutagenesis, Expressing, Incubation

    13) Product Images from "MicroRNA-26a-5p and microRNA-23b-3p up-regulate peroxiredoxin III in acute myeloid leukemia"

    Article Title: MicroRNA-26a-5p and microRNA-23b-3p up-regulate peroxiredoxin III in acute myeloid leukemia

    Journal: Leukemia & Lymphoma

    doi: 10.3109/10428194.2014.924115

    PrxIII is a direct target of both miR-26a-5p and miR-23b-3p. (A) HEK293 cells were transfected with miRNA inhibitors specific for miR-26a-5p, miR-23b-3p and miR-26a-5p + miR-23b-3p. Equivalent amounts (30 μg) of whole-cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with antibodies specific for the indicated proteins. (B) HEK293 cells were transfected with miRNA mimics specific for miR-26a-5p, miR-23b-3p and miR-26a-5p + miR-23b-3p. Equivalent amounts (30 μg) of whole-cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with antibodies specific for the indicated proteins. (C) Compiled data of PrxIII protein analysis from three independent experiments after transfection of miRNAs is shown. Columns, mean; bars, ± SD; ∗ p
    Figure Legend Snippet: PrxIII is a direct target of both miR-26a-5p and miR-23b-3p. (A) HEK293 cells were transfected with miRNA inhibitors specific for miR-26a-5p, miR-23b-3p and miR-26a-5p + miR-23b-3p. Equivalent amounts (30 μg) of whole-cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with antibodies specific for the indicated proteins. (B) HEK293 cells were transfected with miRNA mimics specific for miR-26a-5p, miR-23b-3p and miR-26a-5p + miR-23b-3p. Equivalent amounts (30 μg) of whole-cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with antibodies specific for the indicated proteins. (C) Compiled data of PrxIII protein analysis from three independent experiments after transfection of miRNAs is shown. Columns, mean; bars, ± SD; ∗ p

    Techniques Used: Transfection, SDS Page

    miR-26a-5p and miR-23b-3p decrease levels of ROS. (A) HEK293 and K562 cells transfected with control, miR-mock, miR-26a-5p inhibitor and miR-23b-3p inhibitor were labeled with DCFH-DA and analyzed by flow cytometry. A representative histogram (green, red and blue lines indicate ROS production in transfection of miR-mock, miR-26a-5p inhibitor and miR-23b-3p inhibitor, respectively) is shown. (B) DCFH-DA fluorescence measurements of cellular ROS levels in transfection of miR-mock, miR-26a-5p inhibitor and miR-23b-3p inhibitor in HEK293 or K562 cells. Columns, mean; bars, ± SD; ∗ p
    Figure Legend Snippet: miR-26a-5p and miR-23b-3p decrease levels of ROS. (A) HEK293 and K562 cells transfected with control, miR-mock, miR-26a-5p inhibitor and miR-23b-3p inhibitor were labeled with DCFH-DA and analyzed by flow cytometry. A representative histogram (green, red and blue lines indicate ROS production in transfection of miR-mock, miR-26a-5p inhibitor and miR-23b-3p inhibitor, respectively) is shown. (B) DCFH-DA fluorescence measurements of cellular ROS levels in transfection of miR-mock, miR-26a-5p inhibitor and miR-23b-3p inhibitor in HEK293 or K562 cells. Columns, mean; bars, ± SD; ∗ p

    Techniques Used: Transfection, Labeling, Flow Cytometry, Cytometry, Fluorescence

    14) Product Images from "Mapping of protein phosphatase-6 association with its SAPS domain regulatory subunit using a model of helical repeats"

    Article Title: Mapping of protein phosphatase-6 association with its SAPS domain regulatory subunit using a model of helical repeats

    Journal: BMC Biochemistry

    doi: 10.1186/1471-2091-10-24

    Deletion mapping of PP6c binding to PP6R3 . FLAG-tagged full length PP6R3 (lane 1), or residues 1-355 (lane 2), 1-513 (lane 3) or 512-873 (lane 4) were expressed in HEK293 cells and immunoprecipitated with anti-FLAG antibody. FLAG-tagged proteins (upper panel) and co-immunoprecipitated endogenous PP6c (lower panel) were detected by immunoblotting.
    Figure Legend Snippet: Deletion mapping of PP6c binding to PP6R3 . FLAG-tagged full length PP6R3 (lane 1), or residues 1-355 (lane 2), 1-513 (lane 3) or 512-873 (lane 4) were expressed in HEK293 cells and immunoprecipitated with anti-FLAG antibody. FLAG-tagged proteins (upper panel) and co-immunoprecipitated endogenous PP6c (lower panel) were detected by immunoblotting.

    Techniques Used: Binding Assay, Immunoprecipitation

    Mapping PP6c binding by charge-reversal mutations in PP6R3 . (A) FLAG-tagged full length (FL) PP6R3 (A), and mutants E63-64K (B), D113R (C), E204-205K (D), E259-262K (E) and E204-205-259-262K (F) were expressed in HEK293 cells and immunoprecipitated using immobilized anti-FLAG antibody. Co-precipitated PP6c was quantified by fluorescent immunoblotting and normalized for the amount of FLAG-tagged protein. Results were replicated in 3 independent experiments and plotted as mean +/- SD. (B) Immunoblot of co-precipitated endogenous PP6c from one experiment (upper panel) and the FLAG-tagged PP6R3 proteins (lower panel).
    Figure Legend Snippet: Mapping PP6c binding by charge-reversal mutations in PP6R3 . (A) FLAG-tagged full length (FL) PP6R3 (A), and mutants E63-64K (B), D113R (C), E204-205K (D), E259-262K (E) and E204-205-259-262K (F) were expressed in HEK293 cells and immunoprecipitated using immobilized anti-FLAG antibody. Co-precipitated PP6c was quantified by fluorescent immunoblotting and normalized for the amount of FLAG-tagged protein. Results were replicated in 3 independent experiments and plotted as mean +/- SD. (B) Immunoblot of co-precipitated endogenous PP6c from one experiment (upper panel) and the FLAG-tagged PP6R3 proteins (lower panel).

    Techniques Used: Binding Assay, Immunoprecipitation

    15) Product Images from "Forkhead Transcription Factor FOXO3a Levels Are Increased in Huntington Disease Because of Overactivated Positive Autofeedback Loop *"

    Article Title: Forkhead Transcription Factor FOXO3a Levels Are Increased in Huntington Disease Because of Overactivated Positive Autofeedback Loop *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.612424

    Localization of FOXO3a-like immunoreactive signal in Hdh cells and validation of FOXO3a antibody. A , representative micrographs demonstrating differential distribution of FOXO3a antibody staining in Hdh 7/7 , Hdh 7/109 , and Hdh 109/109 cells. B , immunocytochemical analysis with FOXO3a antibodies in Hdh 7/7 cells left untreated or treated with 1 m m 3-NP for 48 h. C , co-localization of EGFP signal and FOXO3a antibody staining in HEK293 cells transfected with EGFP-FOXO3a encoding construct. In A–C , DNA was counterstained with DAPI. Scale bar , 20 μm. D , Western blot analysis with FOXO3a antibodies of HEK293 cells left untransfected (control) or transfected with Flag-FOXO3a encoding construct, scrambled siRNA, or siRNAs 1 and 2 against FOXO3a . Flag-specific antibodies were used for verification of Flag-FOXO3a expression and tubulin β served as loading control. E , quantification of the data in D . FOXO3a signals were normalized to tubulin β signals. F , RT-qPCR analysis of FOXO3a mRNA levels in siRNAs transfected HEK293 cells performed in parallel with Western blotting. In E and F , the mean results from two independent experiments are shown.
    Figure Legend Snippet: Localization of FOXO3a-like immunoreactive signal in Hdh cells and validation of FOXO3a antibody. A , representative micrographs demonstrating differential distribution of FOXO3a antibody staining in Hdh 7/7 , Hdh 7/109 , and Hdh 109/109 cells. B , immunocytochemical analysis with FOXO3a antibodies in Hdh 7/7 cells left untreated or treated with 1 m m 3-NP for 48 h. C , co-localization of EGFP signal and FOXO3a antibody staining in HEK293 cells transfected with EGFP-FOXO3a encoding construct. In A–C , DNA was counterstained with DAPI. Scale bar , 20 μm. D , Western blot analysis with FOXO3a antibodies of HEK293 cells left untransfected (control) or transfected with Flag-FOXO3a encoding construct, scrambled siRNA, or siRNAs 1 and 2 against FOXO3a . Flag-specific antibodies were used for verification of Flag-FOXO3a expression and tubulin β served as loading control. E , quantification of the data in D . FOXO3a signals were normalized to tubulin β signals. F , RT-qPCR analysis of FOXO3a mRNA levels in siRNAs transfected HEK293 cells performed in parallel with Western blotting. In E and F , the mean results from two independent experiments are shown.

    Techniques Used: Staining, Transfection, Construct, Western Blot, Expressing, Quantitative RT-PCR

    16) Product Images from "Human MAMLD1 Gene Variations Seem Not Sufficient to Explain a 46,XY DSD Phenotype"

    Article Title: Human MAMLD1 Gene Variations Seem Not Sufficient to Explain a 46,XY DSD Phenotype

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0142831

    Effect of MAMLD1 on CYP17A1 promoter and enzyme activities. HEK293 cells or NCI-H295R cells were transiently transfected with MAMLD1 WT and mutant expression vectors. For promoter activation studies, the (-3.7kb) CYP17A1 promoter luciferase reporter construct was co-transfected. A. CYP17A1 promoter activation by MAMLD1 was assessed by the Promega Dual luciferase assay in HEK293 cells. Only for mutant MAMLD1 L210X and L724V an impaired CYP17A1 activation was found. Results are expressed in RLU and represent the mean and SEM of 3 independent experiments performed in duplicate. B. The effect of WT and mutant MAMLD1 on CYP17A1 enzyme activity was assessed in transfected NCI-H295R, MA-10 and HEK293 cells by measuring the conversion of progesterone to 17-hydroxyprogesterone. Steroid production was labeled with [ 14 C]progesterone for 60 min. Steroids were extracted and resolved by thin-layer chromatography, then quantified as % conversion. A representative steroid profile obtained from NCI-H295R cells is shown (n = 2). No effect of MAMLD1 on CYP17A1-hydroxylase activity was detected. P: progesterone; 17OHP: 17-hydroxyprogesterone; RLU: relative light units; Ve: empty vector; WT: wild type; NT: non-transfected; * p ≤0.05.
    Figure Legend Snippet: Effect of MAMLD1 on CYP17A1 promoter and enzyme activities. HEK293 cells or NCI-H295R cells were transiently transfected with MAMLD1 WT and mutant expression vectors. For promoter activation studies, the (-3.7kb) CYP17A1 promoter luciferase reporter construct was co-transfected. A. CYP17A1 promoter activation by MAMLD1 was assessed by the Promega Dual luciferase assay in HEK293 cells. Only for mutant MAMLD1 L210X and L724V an impaired CYP17A1 activation was found. Results are expressed in RLU and represent the mean and SEM of 3 independent experiments performed in duplicate. B. The effect of WT and mutant MAMLD1 on CYP17A1 enzyme activity was assessed in transfected NCI-H295R, MA-10 and HEK293 cells by measuring the conversion of progesterone to 17-hydroxyprogesterone. Steroid production was labeled with [ 14 C]progesterone for 60 min. Steroids were extracted and resolved by thin-layer chromatography, then quantified as % conversion. A representative steroid profile obtained from NCI-H295R cells is shown (n = 2). No effect of MAMLD1 on CYP17A1-hydroxylase activity was detected. P: progesterone; 17OHP: 17-hydroxyprogesterone; RLU: relative light units; Ve: empty vector; WT: wild type; NT: non-transfected; * p ≤0.05.

    Techniques Used: Transfection, Mutagenesis, Expressing, Activation Assay, Luciferase, Construct, Activity Assay, Labeling, Thin Layer Chromatography, Plasmid Preparation

    Transactivation activity of MAMLD1 on the Hes3 promoter. HEK293 cells were transiently transfected with wild-type (WT) and mutant MAMLD1 expression vectors and with a Hes3 promoter luciferase reporter construct. Luciferase activity was measured with the Promega Dual Luciferase assay system. A. Comparison of the newly constructed MAMLD1 WT expression vector (WT (a), NM_005491.4) with the older WT (WT (b)), and ΔE5 (ΔE5 (b)) constructs [ 14 ]. Similar transactivation activity on the Hes3 promoter was found for all constructs. B. Hes3 transactivation by WT and the 11 MAMLD1 mutants was assessed. Only the L210X MAMLD1 mutant showed an impaired activity on the Hes3 promoter. Results are expressed in relative light units (RLU) and represent the mean and SEM of 3 independent experiments performed in duplicate. ΔE5: original WT (b) without exon 5 [ 14 ]; * p ≤0.05.
    Figure Legend Snippet: Transactivation activity of MAMLD1 on the Hes3 promoter. HEK293 cells were transiently transfected with wild-type (WT) and mutant MAMLD1 expression vectors and with a Hes3 promoter luciferase reporter construct. Luciferase activity was measured with the Promega Dual Luciferase assay system. A. Comparison of the newly constructed MAMLD1 WT expression vector (WT (a), NM_005491.4) with the older WT (WT (b)), and ΔE5 (ΔE5 (b)) constructs [ 14 ]. Similar transactivation activity on the Hes3 promoter was found for all constructs. B. Hes3 transactivation by WT and the 11 MAMLD1 mutants was assessed. Only the L210X MAMLD1 mutant showed an impaired activity on the Hes3 promoter. Results are expressed in relative light units (RLU) and represent the mean and SEM of 3 independent experiments performed in duplicate. ΔE5: original WT (b) without exon 5 [ 14 ]; * p ≤0.05.

    Techniques Used: Activity Assay, Transfection, Mutagenesis, Expressing, Luciferase, Construct, Plasmid Preparation

    17) Product Images from "Inflammasome-dependent IL-1β release depends upon membrane permeabilisation"

    Article Title: Inflammasome-dependent IL-1β release depends upon membrane permeabilisation

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2015.176

    Punicalagin does not block NLRP3 or caspase-1 activation. ( a ) Intracellular Ca 2+ rise in mouse BMDMs primed with LPS (1 μ g/ml, 4 h) followed by stimulation with ATP (1 mM, added when indicated with an arrow) in the absence or presence of punicalagin (PUN; 25 μ M). ( b ) Relative intracellular K + concentration of BMDMs treated as in ( a ) with ATP for 30 min. ( c ) Kinetic of net BRET signal for NLRP3 protein expressed in P2X7-HEK293 cells unstimulated or stimulated with ATP (5 mM, added when indicated with an arrow). ( d ) Average quantification (top) and fluorescence microscopy images (bottom) of immortalised ASC-Cherry macrophages containing ASC specks treated as in ( b ); n > 400 cells/condition from 2 independent experiments; scale bar represents 20 μ m. ( e and f ) Immunoblot analysis ( e ) and caspase-1 activity measurements ( f ) of cell lysate and supernatant of BMDMs treated as in ( b ); *** P
    Figure Legend Snippet: Punicalagin does not block NLRP3 or caspase-1 activation. ( a ) Intracellular Ca 2+ rise in mouse BMDMs primed with LPS (1 μ g/ml, 4 h) followed by stimulation with ATP (1 mM, added when indicated with an arrow) in the absence or presence of punicalagin (PUN; 25 μ M). ( b ) Relative intracellular K + concentration of BMDMs treated as in ( a ) with ATP for 30 min. ( c ) Kinetic of net BRET signal for NLRP3 protein expressed in P2X7-HEK293 cells unstimulated or stimulated with ATP (5 mM, added when indicated with an arrow). ( d ) Average quantification (top) and fluorescence microscopy images (bottom) of immortalised ASC-Cherry macrophages containing ASC specks treated as in ( b ); n > 400 cells/condition from 2 independent experiments; scale bar represents 20 μ m. ( e and f ) Immunoblot analysis ( e ) and caspase-1 activity measurements ( f ) of cell lysate and supernatant of BMDMs treated as in ( b ); *** P

    Techniques Used: Blocking Assay, Activation Assay, Concentration Assay, Bioluminescence Resonance Energy Transfer, Fluorescence, Microscopy, Activity Assay

    18) Product Images from "Feedback control of ErbB2 via ERK-mediated phosphorylation of a conserved threonine in the juxtamembrane domain"

    Article Title: Feedback control of ErbB2 via ERK-mediated phosphorylation of a conserved threonine in the juxtamembrane domain

    Journal: Scientific Reports

    doi: 10.1038/srep31502

    Feedback inhibition of ErbB2/ErbB3 via ERK-mediated phosphorylation of ErbB2. ( a ) 293-ErbB2/3 cells were pretreated with trametinib (0.03 μM), and then treated with TPA for 10 min. ( b ) 293-ErbB2/3 cells were pretreated with SCH772984 (0.5 μM), and then treated with TPA for 10 min. ( c ) BT-474 cells were pretreated with U0126 (10 μM), and then treated with TPA for 10 min. ( d ) HEK293 cells were transiently transfected with ErbB2 (WT or T677D) and ErbB3. ( e ) HEK293 cells transiently transfected with ErbB2 (WT or T677A) and ErbB3 were treated with TPA for 10 min. Whole cell lysates were subjected to immunoblotting with antibodies against phospho-ErbB2 (Thr-677) (clone No. 18-4), pY-ErbB2 (Tyr-1196), ErbB2, pY-ErbB3, ErbB3, p-ERK, and Actin. ( f ) Band densities were determined and the level of pY-ErbB2 and pY-ErbB3 were calculated as a ratio against total ErbB2 and ErbB3 quantity, respectively. Values (% of control) represent mean ± S.D. from five independent experiments, *p
    Figure Legend Snippet: Feedback inhibition of ErbB2/ErbB3 via ERK-mediated phosphorylation of ErbB2. ( a ) 293-ErbB2/3 cells were pretreated with trametinib (0.03 μM), and then treated with TPA for 10 min. ( b ) 293-ErbB2/3 cells were pretreated with SCH772984 (0.5 μM), and then treated with TPA for 10 min. ( c ) BT-474 cells were pretreated with U0126 (10 μM), and then treated with TPA for 10 min. ( d ) HEK293 cells were transiently transfected with ErbB2 (WT or T677D) and ErbB3. ( e ) HEK293 cells transiently transfected with ErbB2 (WT or T677A) and ErbB3 were treated with TPA for 10 min. Whole cell lysates were subjected to immunoblotting with antibodies against phospho-ErbB2 (Thr-677) (clone No. 18-4), pY-ErbB2 (Tyr-1196), ErbB2, pY-ErbB3, ErbB3, p-ERK, and Actin. ( f ) Band densities were determined and the level of pY-ErbB2 and pY-ErbB3 were calculated as a ratio against total ErbB2 and ErbB3 quantity, respectively. Values (% of control) represent mean ± S.D. from five independent experiments, *p

    Techniques Used: Inhibition, Transfection

    TPA-induced phosphorylation of ErbB2 at Thr-677. ( a ) Alignment of amino acid sequences around EGFR Thr-669 among four ErbB members is shown. Amino acids corresponding to EGFR Thr-669, ErbB2 Thr-677 and ErbB3 Asp-667 are shown in green. ( b ) HEK293 cells were transiently transfected with expression plasmids for wild type (WT) and Thr-677-Ala mutant (T677A) ErbB2. Twenty-four hours after transfection, cells were treated with TPA for 10 min. ( c ) 293-ErbB2/3 and 293-EGFR cells were treated with TPA for 10 min. The arrowhead shows cross reactivity to EGFR. ( d ) BT-474 cells were treated with TPA for the indicated time. Whole cell lysates were subjected to immunoblotting with antibodies against phospho-ErbB2 (Thr-677) (clones No. 18-1 and 18-4), phospho-ErbB2 (Tyr-1196 and Tyr-1248), ErbB2, phospho-EGFR (Thr-669), EGFR, phospho-ERK, and Actin.
    Figure Legend Snippet: TPA-induced phosphorylation of ErbB2 at Thr-677. ( a ) Alignment of amino acid sequences around EGFR Thr-669 among four ErbB members is shown. Amino acids corresponding to EGFR Thr-669, ErbB2 Thr-677 and ErbB3 Asp-667 are shown in green. ( b ) HEK293 cells were transiently transfected with expression plasmids for wild type (WT) and Thr-677-Ala mutant (T677A) ErbB2. Twenty-four hours after transfection, cells were treated with TPA for 10 min. ( c ) 293-ErbB2/3 and 293-EGFR cells were treated with TPA for 10 min. The arrowhead shows cross reactivity to EGFR. ( d ) BT-474 cells were treated with TPA for the indicated time. Whole cell lysates were subjected to immunoblotting with antibodies against phospho-ErbB2 (Thr-677) (clones No. 18-1 and 18-4), phospho-ErbB2 (Tyr-1196 and Tyr-1248), ErbB2, phospho-EGFR (Thr-669), EGFR, phospho-ERK, and Actin.

    Techniques Used: Transfection, Expressing, Mutagenesis

    Downregulation of ErbB2 and ErbB3 tyrosine phosphorylation. BT-474 and MDA-MB-453 cells were treated with 1 μM lapatinib ( a ) or 100 ng/ml TPA ( b ) for the indicated time. ( c ) BT-474 cells were stimulated with 10 ng/ml EGF, FGF2, or 50 ng/ml HRG for 10 min. ( d , e ) MKN-45 cells and HEK293 cells stably expressing both ErbB2 and ErbB3 (293-ErbB2/3) were treated with TPA for 10 min. Whole cell lysates were subjected to immunoblotting with antibodies against phospho-specific ErbB2 Tyr-1196 (pY-ErbB2), ErbB3 Tyr-1289 (pY-ErbB3), ErbB2, ErbB3, phospho-ERK (pERK), and Actin. The difference of pERK band intensity in each non-treated cells was due to the different exposure time between experiments.
    Figure Legend Snippet: Downregulation of ErbB2 and ErbB3 tyrosine phosphorylation. BT-474 and MDA-MB-453 cells were treated with 1 μM lapatinib ( a ) or 100 ng/ml TPA ( b ) for the indicated time. ( c ) BT-474 cells were stimulated with 10 ng/ml EGF, FGF2, or 50 ng/ml HRG for 10 min. ( d , e ) MKN-45 cells and HEK293 cells stably expressing both ErbB2 and ErbB3 (293-ErbB2/3) were treated with TPA for 10 min. Whole cell lysates were subjected to immunoblotting with antibodies against phospho-specific ErbB2 Tyr-1196 (pY-ErbB2), ErbB3 Tyr-1289 (pY-ErbB3), ErbB2, ErbB3, phospho-ERK (pERK), and Actin. The difference of pERK band intensity in each non-treated cells was due to the different exposure time between experiments.

    Techniques Used: Multiple Displacement Amplification, Stable Transfection, Expressing

    19) Product Images from "Clonal redemption of autoantibodies by somatic hypermutation away from self-reactivity during human immunization"

    Article Title: Clonal redemption of autoantibodies by somatic hypermutation away from self-reactivity during human immunization

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20151978

    Testing polyreactivity of IGHV4-34 antibodies. (A) Flow cytometric evaluation of binding preimmune and mutated IGHV4-34 and negative and low-positive control antibodies mGO53 and eiJB40, respectively, to intact (extracellular) and permeabilized (intracellular) HEK293 cells. Data are representative of two independent experiments. (B) Binding of IGHV4-34 IgG and control antibodies to LPS and dsDNA by ELISA. Data points are the mean and standard deviation of triplicate data points, representative of two independent experiments.
    Figure Legend Snippet: Testing polyreactivity of IGHV4-34 antibodies. (A) Flow cytometric evaluation of binding preimmune and mutated IGHV4-34 and negative and low-positive control antibodies mGO53 and eiJB40, respectively, to intact (extracellular) and permeabilized (intracellular) HEK293 cells. Data are representative of two independent experiments. (B) Binding of IGHV4-34 IgG and control antibodies to LPS and dsDNA by ELISA. Data points are the mean and standard deviation of triplicate data points, representative of two independent experiments.

    Techniques Used: Flow Cytometry, Binding Assay, Positive Control, Enzyme-linked Immunosorbent Assay, Standard Deviation

    20) Product Images from "Rac1 augments Wnt signaling by stimulating β-catenin–lymphoid enhancer factor-1 complex assembly independent of β-catenin nuclear import"

    Article Title: Rac1 augments Wnt signaling by stimulating β-catenin–lymphoid enhancer factor-1 complex assembly independent of β-catenin nuclear import

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.167742

    Activation of Rac1 causes Rac1 – β - catenin complexes to move from the plasma membrane to the nucleus. (A) Left panel: IP of endogenous β-catenin detects association with Rac1 in HEK293 cell lysate; right panel: schematic diagram illustrates the steps involved with the Duolink in situ PLA. The process consists of primary antibodies and PLA probes binding to the target proteins (e.g. β-catenin and Rac1), hybridization and ligation followed by rolling circle amplification. (B) HEK293T cells were stained with primary antibodies for either endogenous total Rac1 or active Rac1 (GTP) and β-catenin, processed for detection of Rac1–β-catenin complexes by PLA technique and imaged using confocal microscopy (see Materials and Methods). Each red dot represents a single interaction between Rac1 and β-catenin. Nuclei were stained with Hoechst (blue) and β-catenin cellular staining is shown in green. Column graph displays the fluorescence intensity of the PLA red dots at the adherens junctions (AJ), nucleus (nuc) or cytosol (cyto) for Rac1(total)–β-catenin and Rac1(GTP)–β-catenin complexes. There was a significant increase in active Rac1–β-catenin complexes in the nucleus, compared with total Rac1–β-catenin complexes, correlating with a reduction in complexes at the adherens junctions. Control images can be found in Fig. S2A . (C) HEK293T and NIH 3T3 cells were transfected with plasmids for GFP-tagged WT (wild-type), T17N or Q61L Rac1 before being stained with anti-GFP and anti-β-catenin antibodies to detect interactions between Rac1 and β-catenin using the PLA. Cells were then stained for β-catenin (green) and DNA (blue), and the PLA-positive red dots each represent an amplified Rac1-GFP/β-catenin interaction complex. For the controls, only one primary antibody (β-catenin or GFP) was added to each well ( Fig. S2B ). The dot plot represents the number of positive nuclear PLA signals per cell for both the single antibody controls (GFP pAb or β-catenin mAb) or both antibodies for each Rac1 construct. The dot plot is representative of two independent experiments each containing 50 cells scored from z -stack confocal microscopy images. (D) Schematic diagram illustrating Rac1–β-catenin complex cellular distribution. When Rac1 is predominantly in its inactive form, Rac1(GDP)–β-catenin complexes are strongly located at the membrane and adherens junctions. Alternatively, when Rac1 is active, Rac1(GTP)–β-catenin complexes displayed a more nuclear-cytoplasmic distribution pattern.
    Figure Legend Snippet: Activation of Rac1 causes Rac1 – β - catenin complexes to move from the plasma membrane to the nucleus. (A) Left panel: IP of endogenous β-catenin detects association with Rac1 in HEK293 cell lysate; right panel: schematic diagram illustrates the steps involved with the Duolink in situ PLA. The process consists of primary antibodies and PLA probes binding to the target proteins (e.g. β-catenin and Rac1), hybridization and ligation followed by rolling circle amplification. (B) HEK293T cells were stained with primary antibodies for either endogenous total Rac1 or active Rac1 (GTP) and β-catenin, processed for detection of Rac1–β-catenin complexes by PLA technique and imaged using confocal microscopy (see Materials and Methods). Each red dot represents a single interaction between Rac1 and β-catenin. Nuclei were stained with Hoechst (blue) and β-catenin cellular staining is shown in green. Column graph displays the fluorescence intensity of the PLA red dots at the adherens junctions (AJ), nucleus (nuc) or cytosol (cyto) for Rac1(total)–β-catenin and Rac1(GTP)–β-catenin complexes. There was a significant increase in active Rac1–β-catenin complexes in the nucleus, compared with total Rac1–β-catenin complexes, correlating with a reduction in complexes at the adherens junctions. Control images can be found in Fig. S2A . (C) HEK293T and NIH 3T3 cells were transfected with plasmids for GFP-tagged WT (wild-type), T17N or Q61L Rac1 before being stained with anti-GFP and anti-β-catenin antibodies to detect interactions between Rac1 and β-catenin using the PLA. Cells were then stained for β-catenin (green) and DNA (blue), and the PLA-positive red dots each represent an amplified Rac1-GFP/β-catenin interaction complex. For the controls, only one primary antibody (β-catenin or GFP) was added to each well ( Fig. S2B ). The dot plot represents the number of positive nuclear PLA signals per cell for both the single antibody controls (GFP pAb or β-catenin mAb) or both antibodies for each Rac1 construct. The dot plot is representative of two independent experiments each containing 50 cells scored from z -stack confocal microscopy images. (D) Schematic diagram illustrating Rac1–β-catenin complex cellular distribution. When Rac1 is predominantly in its inactive form, Rac1(GDP)–β-catenin complexes are strongly located at the membrane and adherens junctions. Alternatively, when Rac1 is active, Rac1(GTP)–β-catenin complexes displayed a more nuclear-cytoplasmic distribution pattern.

    Techniques Used: Activation Assay, In Situ, Proximity Ligation Assay, Binding Assay, Hybridization, Ligation, Amplification, Staining, Confocal Microscopy, Fluorescence, Transfection, Construct

    Rac1 stimulates the formation of endogenous β - catenin – LEF-1 complexes. (A) HEK293T cells were stained with primary antibodies and processed for detection of interactions between endogenous β-catenin and LEF-1 using the proximity ligation assay (PLA) Duolink. As shown in the cell images, cells were either transfected with Rac1 constructs (WT, T17N or Q61L) or treated with Wnt3a conditioned media, 40 mM LiCl, 50 µM NSC23766 or 50 µM NSC23766+Wnt3a for 6 h before being fixed and subjected to Duolink assay. The assay was performed using both anti-β-catenin and anti-LEF-1 antibodies (see Materials and Methods for details). For the control wells, only one primary antibody (β-catenin or LEF-1) was added. Cells were then stained for β-catenin (green) and the PLA-positive red dots detected each represent an amplified β-catenin/LEF-1 interaction complex. (B) The PLA signals were counted and the number of dots in the nucleus per cell from single slice confocal images is presented in the plot. One hundred nuclei from two independent experiments were scored. (C) GFP, GFP-β-catenin and HA-tagged LEF1 were transiently expressed in HEK293 cells and total lysates used for IP by anti-HA antibody. The IP shows detection of LEF-1–β-catenin complexes in cells untreated or treated for 6 h with drug. (D) Summary diagram showing that the interaction between β-catenin and LEF-1 is stimulated in the presence of Wnt and active Rac1, and inhibited in the presence of inactive Rac1.
    Figure Legend Snippet: Rac1 stimulates the formation of endogenous β - catenin – LEF-1 complexes. (A) HEK293T cells were stained with primary antibodies and processed for detection of interactions between endogenous β-catenin and LEF-1 using the proximity ligation assay (PLA) Duolink. As shown in the cell images, cells were either transfected with Rac1 constructs (WT, T17N or Q61L) or treated with Wnt3a conditioned media, 40 mM LiCl, 50 µM NSC23766 or 50 µM NSC23766+Wnt3a for 6 h before being fixed and subjected to Duolink assay. The assay was performed using both anti-β-catenin and anti-LEF-1 antibodies (see Materials and Methods for details). For the control wells, only one primary antibody (β-catenin or LEF-1) was added. Cells were then stained for β-catenin (green) and the PLA-positive red dots detected each represent an amplified β-catenin/LEF-1 interaction complex. (B) The PLA signals were counted and the number of dots in the nucleus per cell from single slice confocal images is presented in the plot. One hundred nuclei from two independent experiments were scored. (C) GFP, GFP-β-catenin and HA-tagged LEF1 were transiently expressed in HEK293 cells and total lysates used for IP by anti-HA antibody. The IP shows detection of LEF-1–β-catenin complexes in cells untreated or treated for 6 h with drug. (D) Summary diagram showing that the interaction between β-catenin and LEF-1 is stimulated in the presence of Wnt and active Rac1, and inhibited in the presence of inactive Rac1.

    Techniques Used: Staining, Proximity Ligation Assay, Transfection, Construct, Amplification

    21) Product Images from "Endothelial Cell-Specific Molecule 2 (ECSM2) Localizes to Cell-Cell Junctions and Modulates bFGF-Directed Cell Migration via the ERK-FAK Pathway"

    Article Title: Endothelial Cell-Specific Molecule 2 (ECSM2) Localizes to Cell-Cell Junctions and Modulates bFGF-Directed Cell Migration via the ERK-FAK Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0021482

    Generation of rabbit anti-ECSM2 monoclonal antibody (RabMAb) and characterization of endogenous ECSM2. (A) Diagram of human ECSM2 constructs used in generation and screening of anti-ECSM2 antibodies. SP, signal peptide; TM, transmembrane domain; ECD, extracellular domain; ICD, intracellular domain. (B) Anti-ECSM2 antiserum specifically detects His-ICD. Purified GST-ICD proteins were used to immunize the rabbit to generate polyclonal antibody (pAb) and monoclonal antibody (RabMAb) sequentially as detailed in Methods . Antiserum (bleed containing pAb) was collected and used for immunoblotting. His-Ctrl: His-tagged non-related protein (control). (C) Anti-ECSM2 pAb recognizes ECSM2-GFP but not GFP. Protein extracts from the HEK293 cells overexpressing human ECSM2-GFP or GFP alone were used for immunoblotting. (D) Anti-ECSM2 RabMAb specifically detects endogenous ECSM2 in human ECs. Protein extracts from human EC lines (HUVEC and HDMVEC), mouse endothelial MS1 cells, and non-EC lines (MCF-7, DU145 and HEK293) were analyzed by immunoblotting with anti-ECSM2 RabMAb subclone 71-1 (hybridoma supernatant) or anti-β-actin (loading control). (E) Quantitative RT-PCR analysis measuring the ECSM2 mRNA levels in HUVEC and MS1 cells, normalized to human and mouse GAPDH, respectively. Data are mean±SEM (n = 6). **, P
    Figure Legend Snippet: Generation of rabbit anti-ECSM2 monoclonal antibody (RabMAb) and characterization of endogenous ECSM2. (A) Diagram of human ECSM2 constructs used in generation and screening of anti-ECSM2 antibodies. SP, signal peptide; TM, transmembrane domain; ECD, extracellular domain; ICD, intracellular domain. (B) Anti-ECSM2 antiserum specifically detects His-ICD. Purified GST-ICD proteins were used to immunize the rabbit to generate polyclonal antibody (pAb) and monoclonal antibody (RabMAb) sequentially as detailed in Methods . Antiserum (bleed containing pAb) was collected and used for immunoblotting. His-Ctrl: His-tagged non-related protein (control). (C) Anti-ECSM2 pAb recognizes ECSM2-GFP but not GFP. Protein extracts from the HEK293 cells overexpressing human ECSM2-GFP or GFP alone were used for immunoblotting. (D) Anti-ECSM2 RabMAb specifically detects endogenous ECSM2 in human ECs. Protein extracts from human EC lines (HUVEC and HDMVEC), mouse endothelial MS1 cells, and non-EC lines (MCF-7, DU145 and HEK293) were analyzed by immunoblotting with anti-ECSM2 RabMAb subclone 71-1 (hybridoma supernatant) or anti-β-actin (loading control). (E) Quantitative RT-PCR analysis measuring the ECSM2 mRNA levels in HUVEC and MS1 cells, normalized to human and mouse GAPDH, respectively. Data are mean±SEM (n = 6). **, P

    Techniques Used: Construct, Purification, Quantitative RT-PCR

    Characterization of ECSM2 proteins by enzymatic deglycosylation. (A) Glycosylation of endogenous ECSM2. HUVEC lysates were immunoprecipitated with anti-ECSM2 RabMAb (lanes 2 and 3) or rabbit IgG as a control (lane 1). Samples were treated with (+) or without (-) glycosidase mix, as detailed in Methods , resolved by SDS-PAGE, and immunoblotted with anti-ECSM2 RabMAb. Positions of glycosylated (mature) ECSM2, deglycosylated ECSM2, and IgG are indicated. (B) Glycosylation of ECSM2-FLAG. HEK293 cells stably expressing mouse ECSM2-FLAG were lysed and cell lysates were directly subjected to enzymatic deglycosylation reactions as described in Methods . Samples were analyzed by immunoblotting with anti-FLAG M2 mAb (lanes 1 and 2) and anti-ECSM2 RabMAb (lanes 3 and 4), respectively. Positions of glycosylated (mature) ECSM2-FLAG, and deglycosylated ECSM2-FLAG are indicated.
    Figure Legend Snippet: Characterization of ECSM2 proteins by enzymatic deglycosylation. (A) Glycosylation of endogenous ECSM2. HUVEC lysates were immunoprecipitated with anti-ECSM2 RabMAb (lanes 2 and 3) or rabbit IgG as a control (lane 1). Samples were treated with (+) or without (-) glycosidase mix, as detailed in Methods , resolved by SDS-PAGE, and immunoblotted with anti-ECSM2 RabMAb. Positions of glycosylated (mature) ECSM2, deglycosylated ECSM2, and IgG are indicated. (B) Glycosylation of ECSM2-FLAG. HEK293 cells stably expressing mouse ECSM2-FLAG were lysed and cell lysates were directly subjected to enzymatic deglycosylation reactions as described in Methods . Samples were analyzed by immunoblotting with anti-FLAG M2 mAb (lanes 1 and 2) and anti-ECSM2 RabMAb (lanes 3 and 4), respectively. Positions of glycosylated (mature) ECSM2-FLAG, and deglycosylated ECSM2-FLAG are indicated.

    Techniques Used: Immunoprecipitation, SDS Page, Stable Transfection, Expressing

    Overexpression of ECSM2 promotes cell aggregation. (A) HEK293 cells stably expressing GFP (control), human (h) or mouse (m) ECSM2-GFP were used for cell aggregation assays, as described in Methods . Representative images captured by an inverted phase contrast microscope (magnification: ×10) are shown. Scale bar, 200 µm. (B and C) The aggregation indexes for HEK293 stably expressing GFP, human (h) or mouse (m) ECSM2-GFP (B), and MS1 stably expressing GFP or human ECSM2-GFP (C) were calculated and plotted. Data are mean±SEM (n = 8). **, P
    Figure Legend Snippet: Overexpression of ECSM2 promotes cell aggregation. (A) HEK293 cells stably expressing GFP (control), human (h) or mouse (m) ECSM2-GFP were used for cell aggregation assays, as described in Methods . Representative images captured by an inverted phase contrast microscope (magnification: ×10) are shown. Scale bar, 200 µm. (B and C) The aggregation indexes for HEK293 stably expressing GFP, human (h) or mouse (m) ECSM2-GFP (B), and MS1 stably expressing GFP or human ECSM2-GFP (C) were calculated and plotted. Data are mean±SEM (n = 8). **, P

    Techniques Used: Over Expression, Stable Transfection, Expressing, Microscopy

    22) Product Images from "Cadmium Induces Transcription Independently of Intracellular Calcium Mobilization"

    Article Title: Cadmium Induces Transcription Independently of Intracellular Calcium Mobilization

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0020542

    Effects of cadmium and thapsigargin on transcription. Total RNA was isolated from HEK293::YC3.60 cells exposed to either 1 µM ( square ) or 30 µM ( triangle ) cadmium, or 2 µM thapsigargin ( circle ) for 1, 4, or 24 h. Steady-state mRNA levels of mt-1, c-fos, and grp-78 were measured using qRT-PCR. All measurements were normalized to mRNA levels of actin. Fold change was normalized to mRNA levels observed in control cells. Results were mean log 2 fold change ± SEM ( n = 3) and were analyzed by one-way ANOVA followed by Dunnett's post-test; single (*) and double (**) asterisks indicate significant differences from controls at p
    Figure Legend Snippet: Effects of cadmium and thapsigargin on transcription. Total RNA was isolated from HEK293::YC3.60 cells exposed to either 1 µM ( square ) or 30 µM ( triangle ) cadmium, or 2 µM thapsigargin ( circle ) for 1, 4, or 24 h. Steady-state mRNA levels of mt-1, c-fos, and grp-78 were measured using qRT-PCR. All measurements were normalized to mRNA levels of actin. Fold change was normalized to mRNA levels observed in control cells. Results were mean log 2 fold change ± SEM ( n = 3) and were analyzed by one-way ANOVA followed by Dunnett's post-test; single (*) and double (**) asterisks indicate significant differences from controls at p

    Techniques Used: Isolation, Quantitative RT-PCR

    Effect of cadmium on YC3.60 fluorescence in HEK293::YC3.60 cells. Traces represent changes in YC3.60 fluorescence ratios following ionomycin exposure in calcium-free HBSS. At ∼15 min, the medium was supplemented with either 2 mM calcium ( , black line ) or 2 mM cadmium ( , red line ). Traces are representative of typical responses observed in four independent experiments.
    Figure Legend Snippet: Effect of cadmium on YC3.60 fluorescence in HEK293::YC3.60 cells. Traces represent changes in YC3.60 fluorescence ratios following ionomycin exposure in calcium-free HBSS. At ∼15 min, the medium was supplemented with either 2 mM calcium ( , black line ) or 2 mM cadmium ( , red line ). Traces are representative of typical responses observed in four independent experiments.

    Techniques Used: Fluorescence

    Cadmium uptake and cell viability in HEK 293::YC3.60 cells. A , Fura-5F loaded cells were incubated with ionomycin for 10 min to deplete intracellular calcium stores and then 2 mM cadmium in calcium-free HBSS was added to the medium ( solid line ). In the experiment represented by the dashed line , similar conditions were used except cells were not exposed to ionomycin prior to cadmium addition. Traces are representative of typical responses observed in at least three independent experiments. B , HEK293::YC3.60 cells were exposed to 0, 1, 3, 10, and 30 µM cadmium for 4 ( closed circle ) and 24 ( open circle ) h. Following metal exposure, cells were incubated with fura-5F and then fluorescence ratios were determined. Asterisks indicate a significant (p
    Figure Legend Snippet: Cadmium uptake and cell viability in HEK 293::YC3.60 cells. A , Fura-5F loaded cells were incubated with ionomycin for 10 min to deplete intracellular calcium stores and then 2 mM cadmium in calcium-free HBSS was added to the medium ( solid line ). In the experiment represented by the dashed line , similar conditions were used except cells were not exposed to ionomycin prior to cadmium addition. Traces are representative of typical responses observed in at least three independent experiments. B , HEK293::YC3.60 cells were exposed to 0, 1, 3, 10, and 30 µM cadmium for 4 ( closed circle ) and 24 ( open circle ) h. Following metal exposure, cells were incubated with fura-5F and then fluorescence ratios were determined. Asterisks indicate a significant (p

    Techniques Used: Incubation, Fluorescence

    Venn diagram illustrating genes whose steady-state levels of expression change in HEK293::YC3.60 cells following exposure to 1 or 30 µM cadmium for 4 or 24 h, or 2 µM thapsigargin for 4 h. The identity and description of the eleven common genes are presented in Table 4 .
    Figure Legend Snippet: Venn diagram illustrating genes whose steady-state levels of expression change in HEK293::YC3.60 cells following exposure to 1 or 30 µM cadmium for 4 or 24 h, or 2 µM thapsigargin for 4 h. The identity and description of the eleven common genes are presented in Table 4 .

    Techniques Used: Expressing

    panel A , Traces represent [Ca 2+ ] i measured in control HEK293::YC3.60 cells ( black line ) or cells following exposure to 1 µM cadmium for 4 hr ( red line ). The traces are representative of typical responses observed in at least three independent experiments. panel B, Means of the peak values in thapsigargin response following exposure to 1 µM cadmium for 4 hr ( gray bar ) or non-cadmium treated ( black bar ). Data were expressed as the mean ± SEM and were analyzed by an unpaired t-test . There were no significant differences between control and the cadmium exposed groups. panel C , Traces represent [Ca 2+ ] i measured in HEK 293::YC3.60 cells under control conditions ( black line ) or following a 4 h exposure to 1 µM cadmium ( red line ). Following incubation with cadmium, cells were treated with an ionomycin-BAPTA solution in calcium-free HBSS. The traces were representative of typical responses observed in at least three independent experiments. panel D , Mean peak values of ionomycin responses in HEK293::YC3.60 cells following exposure to 0, 1, 3, 10, and 30 µM cadmium for 4 h ( black bar ) or 24 h ( gray bar ). Data were expressed as the mean ± SEM and were analyzed by one-way ANOVA followed by Dunnett's post-test. Asterisks (*) indicate significant difference (p
    Figure Legend Snippet: panel A , Traces represent [Ca 2+ ] i measured in control HEK293::YC3.60 cells ( black line ) or cells following exposure to 1 µM cadmium for 4 hr ( red line ). The traces are representative of typical responses observed in at least three independent experiments. panel B, Means of the peak values in thapsigargin response following exposure to 1 µM cadmium for 4 hr ( gray bar ) or non-cadmium treated ( black bar ). Data were expressed as the mean ± SEM and were analyzed by an unpaired t-test . There were no significant differences between control and the cadmium exposed groups. panel C , Traces represent [Ca 2+ ] i measured in HEK 293::YC3.60 cells under control conditions ( black line ) or following a 4 h exposure to 1 µM cadmium ( red line ). Following incubation with cadmium, cells were treated with an ionomycin-BAPTA solution in calcium-free HBSS. The traces were representative of typical responses observed in at least three independent experiments. panel D , Mean peak values of ionomycin responses in HEK293::YC3.60 cells following exposure to 0, 1, 3, 10, and 30 µM cadmium for 4 h ( black bar ) or 24 h ( gray bar ). Data were expressed as the mean ± SEM and were analyzed by one-way ANOVA followed by Dunnett's post-test. Asterisks (*) indicate significant difference (p

    Techniques Used: Incubation

    23) Product Images from "MYCT1-TV, A Novel MYCT1 Transcript, Is Regulated by c-Myc and May Participate in Laryngeal Carcinogenesis"

    Article Title: MYCT1-TV, A Novel MYCT1 Transcript, Is Regulated by c-Myc and May Participate in Laryngeal Carcinogenesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025648

    The transcriptional activity of the mutant MYCT1-TV core promoter. Promoter constructs pGL3-Basic, P852-mutA, P852-mutB and P852-mutAB were cotransfected with pRL-TK into Hep2 (black bars) or HEK293 (gray bars) cells, respectively by Lipofectamine 2000 Reagent. Luciferase activity was measured at 48 h after transfection (**, p
    Figure Legend Snippet: The transcriptional activity of the mutant MYCT1-TV core promoter. Promoter constructs pGL3-Basic, P852-mutA, P852-mutB and P852-mutAB were cotransfected with pRL-TK into Hep2 (black bars) or HEK293 (gray bars) cells, respectively by Lipofectamine 2000 Reagent. Luciferase activity was measured at 48 h after transfection (**, p

    Techniques Used: Activity Assay, Mutagenesis, Construct, Luciferase, Transfection

    Identification and functional characterization of human MYCT1-TV promoter. A. Nucleotide sequence of the promoter region (from −981 to +52) of human MYCT1-TV . The putative binding sites for transcriptional factors are underlined and markded as E-box A and B. The transcriptional start site is marked in bold and italic at −140 bp. The start nucleotide of the published MYCT1 mRNA sequence is marked in bold at −152 bp. The positions of putative core promoter sequences are marked with gray color. The numbering of the nucleotides starts at initiator codon ATG (+1) which are boxed. B. Analysis of human MYCT1-TV promoter activities detected by luciferase assay. Hep2 (black bars) or HEK293 (gray bars) cells are transiently transfected with 0.8 µg of the deletion constructs together with 16 ng pRL-TK. The relative activities of a series of deletion constructs are determined by luciferase assay (**, p
    Figure Legend Snippet: Identification and functional characterization of human MYCT1-TV promoter. A. Nucleotide sequence of the promoter region (from −981 to +52) of human MYCT1-TV . The putative binding sites for transcriptional factors are underlined and markded as E-box A and B. The transcriptional start site is marked in bold and italic at −140 bp. The start nucleotide of the published MYCT1 mRNA sequence is marked in bold at −152 bp. The positions of putative core promoter sequences are marked with gray color. The numbering of the nucleotides starts at initiator codon ATG (+1) which are boxed. B. Analysis of human MYCT1-TV promoter activities detected by luciferase assay. Hep2 (black bars) or HEK293 (gray bars) cells are transiently transfected with 0.8 µg of the deletion constructs together with 16 ng pRL-TK. The relative activities of a series of deletion constructs are determined by luciferase assay (**, p

    Techniques Used: Functional Assay, Sequencing, Binding Assay, Luciferase, Transfection, Construct

    Function of MYCT1-TV and MYCT1 in Hep2 and HEK293 cells. The proliferation of Hep2 and HEK293 cells. B. The apoptosis of Hep2 and HEK293 cells. C. The invasive ability of Hep2 and HEK293 cells. Blank, control cells before transfection; GFP, control cells after transfection with GFP only; MYCT1-TV, cells transfected with MYCT1-TV-GFP; MYCT1, cells transfected with MYCT1-GFP.
    Figure Legend Snippet: Function of MYCT1-TV and MYCT1 in Hep2 and HEK293 cells. The proliferation of Hep2 and HEK293 cells. B. The apoptosis of Hep2 and HEK293 cells. C. The invasive ability of Hep2 and HEK293 cells. Blank, control cells before transfection; GFP, control cells after transfection with GFP only; MYCT1-TV, cells transfected with MYCT1-TV-GFP; MYCT1, cells transfected with MYCT1-GFP.

    Techniques Used: Transfection

    A role for E-box sites in basal human MTCT1-TV promoter activity. A. Binding of MYCT1-TV E-box sites to c-Myc in vitro detected by EMSAs. The symbol “ * ” means the oligonucleotides labled by biotin. Lanes 1 to 10 stand for the results from Hep2 cells and Lanes 11 to 20 the results from HEK293 cells. Lanes 1, 6, 11 and 16 represent biotin-labled oligonucleotides. Lanes 2, 7, 13 and 18 represent each probe incubated with nuclear extracts. Lanes 3, 8, 14 and 19 represent each probe incubated with a 100-fold excess of the unlabeled competitor oligonucleotides. Lanes 4, 9, 15 and 20 represent each probe incubated with a 100-fold excess of the unlabeled mutant competitor oligonucleotides. Lanes 5, 10, 12 and 17 represent the EMSA results in the presence of anti-c-Myc antibody. The experiments were repeated three times. B. Binding of E-box sites to c-Myc in vivo detected by ChIP. The Input lanes correspond to PCR products derived from chromatin prior to immunoprecipitation. The IgG lanes correspond to PCR products containing chromatin immunoprecipitated with antibodies against control IgG. The c-Myc lanes correspond to PCR products containing chromatin immunoprecipitated with antibodies against c-Myc. M indicates DNA 2000 marker. The 242 bp PCR product of c-Myc A or the 215 bp PCR product of c-Myc B is obtained corresponding to the sequence either E-box A or B binding site of the MYCT1-TV promoter. Results of Hep2 cells shown in the left figure are in line with those of HEK293 cells in the right one.
    Figure Legend Snippet: A role for E-box sites in basal human MTCT1-TV promoter activity. A. Binding of MYCT1-TV E-box sites to c-Myc in vitro detected by EMSAs. The symbol “ * ” means the oligonucleotides labled by biotin. Lanes 1 to 10 stand for the results from Hep2 cells and Lanes 11 to 20 the results from HEK293 cells. Lanes 1, 6, 11 and 16 represent biotin-labled oligonucleotides. Lanes 2, 7, 13 and 18 represent each probe incubated with nuclear extracts. Lanes 3, 8, 14 and 19 represent each probe incubated with a 100-fold excess of the unlabeled competitor oligonucleotides. Lanes 4, 9, 15 and 20 represent each probe incubated with a 100-fold excess of the unlabeled mutant competitor oligonucleotides. Lanes 5, 10, 12 and 17 represent the EMSA results in the presence of anti-c-Myc antibody. The experiments were repeated three times. B. Binding of E-box sites to c-Myc in vivo detected by ChIP. The Input lanes correspond to PCR products derived from chromatin prior to immunoprecipitation. The IgG lanes correspond to PCR products containing chromatin immunoprecipitated with antibodies against control IgG. The c-Myc lanes correspond to PCR products containing chromatin immunoprecipitated with antibodies against c-Myc. M indicates DNA 2000 marker. The 242 bp PCR product of c-Myc A or the 215 bp PCR product of c-Myc B is obtained corresponding to the sequence either E-box A or B binding site of the MYCT1-TV promoter. Results of Hep2 cells shown in the left figure are in line with those of HEK293 cells in the right one.

    Techniques Used: Activity Assay, Binding Assay, In Vitro, Incubation, Mutagenesis, In Vivo, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Derivative Assay, Immunoprecipitation, Marker, Sequencing

    Transfection of MYCT1-TV and MYCT1 in Hep2 and HEK293 cells. A. mRNA levels of MYCT1-TV/MYCT1 in Hep2 and HEK293 cells transfected with MYCT1-TV-GFP/MYCT1-GFP. PCR produce a 929 bp DNA fragment for MYCT1-TV , a 726 bp DNA fragment for MYCT1 and a 511 bp DNA fragment for β-actin. In Hep2 cells, the gray-scale ratios of MYCT1-TV to β-actin mRNA levels (black bars) in blank, GFP and MYCT1-TV-GFP groups are 0.4735±0.0335, 0.4315±0.0303 and 23.5188±2.0896, and the gray-scale ratios of MYCT1 to β-actin mRNA levels (gray bars) in blank, GFP and MYCT1-GFP groups are 0.4157±0.1080, 0.4242±0.0658 and 25.7520±1.3244, respectively. In HEK293 cells, the counterpart gray-scale ratios of MYCT1-TV to β-actin mRNA levels are 1.3071±0.2223, 1.2523±0.1002 and 32.0339±2.2903, and the counterpart gray-scale ratios of MYCT1 to β-actin mRNA levels are 0.9950±0.0725, 1.1448±0.1346 and 31.5161±1.9808, respectively. B. Transfection efficiency and expression of MYCT1-TV/MYCT1 protein in Hep2 and HEK293 cells. Transfection efficiency and expression of MYCT1-TV/MYCT1 protein in GFP, MYCT1-TV-GFP and MYCT1-GFP groups are revealed by contrast and fluorescence microscopy under the same phase. M, DNA marker; blank, control cells before transfection; GFP, control cells after transfection with GFP only; MYCT1-TV-GFP, cells transfected with MYCT1-TV-GFP; MYCT1-GFP, cells transfected with MYCT1-GFP (**, p
    Figure Legend Snippet: Transfection of MYCT1-TV and MYCT1 in Hep2 and HEK293 cells. A. mRNA levels of MYCT1-TV/MYCT1 in Hep2 and HEK293 cells transfected with MYCT1-TV-GFP/MYCT1-GFP. PCR produce a 929 bp DNA fragment for MYCT1-TV , a 726 bp DNA fragment for MYCT1 and a 511 bp DNA fragment for β-actin. In Hep2 cells, the gray-scale ratios of MYCT1-TV to β-actin mRNA levels (black bars) in blank, GFP and MYCT1-TV-GFP groups are 0.4735±0.0335, 0.4315±0.0303 and 23.5188±2.0896, and the gray-scale ratios of MYCT1 to β-actin mRNA levels (gray bars) in blank, GFP and MYCT1-GFP groups are 0.4157±0.1080, 0.4242±0.0658 and 25.7520±1.3244, respectively. In HEK293 cells, the counterpart gray-scale ratios of MYCT1-TV to β-actin mRNA levels are 1.3071±0.2223, 1.2523±0.1002 and 32.0339±2.2903, and the counterpart gray-scale ratios of MYCT1 to β-actin mRNA levels are 0.9950±0.0725, 1.1448±0.1346 and 31.5161±1.9808, respectively. B. Transfection efficiency and expression of MYCT1-TV/MYCT1 protein in Hep2 and HEK293 cells. Transfection efficiency and expression of MYCT1-TV/MYCT1 protein in GFP, MYCT1-TV-GFP and MYCT1-GFP groups are revealed by contrast and fluorescence microscopy under the same phase. M, DNA marker; blank, control cells before transfection; GFP, control cells after transfection with GFP only; MYCT1-TV-GFP, cells transfected with MYCT1-TV-GFP; MYCT1-GFP, cells transfected with MYCT1-GFP (**, p

    Techniques Used: Transfection, Polymerase Chain Reaction, Expressing, Fluorescence, Microscopy, Marker

    Silencing of endogenous c-Myc reduces MYCT1-TV promoter activity. A. Detecting of c-Myc expression level in cells by qRT-PCR. The expression of GAPDH was used as an internal control. Hundred nanogram of no siRNA (Mock), nonspecific control siRNA (NC) or c-Myc specific siRNA (c-Myc) were transfected into Hep2 (black bars) or HEK293 (gray bars) cells. B. Luciferase activity of MYCT1-TV promoter after transfected with c-Myc siRNA. Luciferase activity was measured in Hep2 (black bars) or HEK293 (gray bars) extracts 48 h after transfection. pGL3-Basic, cells cotransfected with pGL3-Basic and pRL-TK; P852, cells cotransfected with P852 and pRL-TK; P852+NC, cells cotransfected with P852, NC siRNA and pRL-TK; P852+c-Myc, cells cotransfected with P852, c-Myc siRNA and pRL-TK. Data are indicated as the means ± SEM of three independent experiments.
    Figure Legend Snippet: Silencing of endogenous c-Myc reduces MYCT1-TV promoter activity. A. Detecting of c-Myc expression level in cells by qRT-PCR. The expression of GAPDH was used as an internal control. Hundred nanogram of no siRNA (Mock), nonspecific control siRNA (NC) or c-Myc specific siRNA (c-Myc) were transfected into Hep2 (black bars) or HEK293 (gray bars) cells. B. Luciferase activity of MYCT1-TV promoter after transfected with c-Myc siRNA. Luciferase activity was measured in Hep2 (black bars) or HEK293 (gray bars) extracts 48 h after transfection. pGL3-Basic, cells cotransfected with pGL3-Basic and pRL-TK; P852, cells cotransfected with P852 and pRL-TK; P852+NC, cells cotransfected with P852, NC siRNA and pRL-TK; P852+c-Myc, cells cotransfected with P852, c-Myc siRNA and pRL-TK. Data are indicated as the means ± SEM of three independent experiments.

    Techniques Used: Activity Assay, Expressing, Quantitative RT-PCR, Transfection, Luciferase

    Expression of MYCT1-TV and MYCT1 in human cells and tissues. A. MYCT1-TV mRNA levels in human cells. The gray-scale ratios of MYCT1-TV to β-actin mRNA levels in Hep2, HeLa, BGC823, SGC7901, Bel7402, GES1, HEK293, human blood and MKN1 cells are 0.2890±0.0521, 0.3113±0.0138, 0.2985±0.0130, 0.2964±0.0427, 0.3512±0.0407, 1.0522±0.0808, 1.1159±0.1467, 1.1641±0.0665 and 0.2348±0.0147, respectively. B. MYCT1-TV and MYCT1 mRNA levels in LSCC and paired adjacent normal laryngeal tissues. PCR produce a 929 bp DNA fragment for MYCT1-TV , a 726 bp DNA fragment for MYCT1 and a 511 bp DNA fragment for β-actin. The gray-scale ratios of MYCT1-TV to β-actin mRNA levels (black bars) in LSCC and paired adjacent normal laryngeal tissues are 0.4172±0.0324 and 0.8073±0.0478, and the counterpart gray-scale ratios of MYCT1 to β-actin mRNA levels (gray bars) are 0.4304±0.0304 and 0.8416±0.0499. M, T and R indicate DNA marker, LSCC tumor tissue and paired adjacent normal tissue, respectively.
    Figure Legend Snippet: Expression of MYCT1-TV and MYCT1 in human cells and tissues. A. MYCT1-TV mRNA levels in human cells. The gray-scale ratios of MYCT1-TV to β-actin mRNA levels in Hep2, HeLa, BGC823, SGC7901, Bel7402, GES1, HEK293, human blood and MKN1 cells are 0.2890±0.0521, 0.3113±0.0138, 0.2985±0.0130, 0.2964±0.0427, 0.3512±0.0407, 1.0522±0.0808, 1.1159±0.1467, 1.1641±0.0665 and 0.2348±0.0147, respectively. B. MYCT1-TV and MYCT1 mRNA levels in LSCC and paired adjacent normal laryngeal tissues. PCR produce a 929 bp DNA fragment for MYCT1-TV , a 726 bp DNA fragment for MYCT1 and a 511 bp DNA fragment for β-actin. The gray-scale ratios of MYCT1-TV to β-actin mRNA levels (black bars) in LSCC and paired adjacent normal laryngeal tissues are 0.4172±0.0324 and 0.8073±0.0478, and the counterpart gray-scale ratios of MYCT1 to β-actin mRNA levels (gray bars) are 0.4304±0.0304 and 0.8416±0.0499. M, T and R indicate DNA marker, LSCC tumor tissue and paired adjacent normal tissue, respectively.

    Techniques Used: Expressing, Polymerase Chain Reaction, Marker

    24) Product Images from "The SARS-Coronavirus-Host Interactome: Identification of Cyclophilins as Target for Pan-Coronavirus Inhibitors"

    Article Title: The SARS-Coronavirus-Host Interactome: Identification of Cyclophilins as Target for Pan-Coronavirus Inhibitors

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002331

    SARS-CoV Nsp1 full length (Nsp1fl) induces NFAT-regulated gene expression in vitro independently of the NFAT molecular species, and the calcipressin RCAN3 extenuates the effect. HEK293 cells were transiently cotransfected with NFAT reporter plasmid (NFAT luc) and expression plasmids encoding NFAT3, Calcineurin (CnA) and SARS-CoV Nsp1fl ( A ). RCAN3 was additionally expressed in ( B ). In ( C ) and ( D ), NFAT1 and NFAT2 species were expressed instead of NFAT3, respectively. The respective empty plasmid vector DNA was added to each individual transfection setup in order to obtain identical DNA concentrations. After transfection cells were cultured in absence or presence of the calcineurin stimulators PMA and ionomycin (PMA/Io.) and the NFAT-pathway inhibitor Cyclosporin A (CspA). ** P
    Figure Legend Snippet: SARS-CoV Nsp1 full length (Nsp1fl) induces NFAT-regulated gene expression in vitro independently of the NFAT molecular species, and the calcipressin RCAN3 extenuates the effect. HEK293 cells were transiently cotransfected with NFAT reporter plasmid (NFAT luc) and expression plasmids encoding NFAT3, Calcineurin (CnA) and SARS-CoV Nsp1fl ( A ). RCAN3 was additionally expressed in ( B ). In ( C ) and ( D ), NFAT1 and NFAT2 species were expressed instead of NFAT3, respectively. The respective empty plasmid vector DNA was added to each individual transfection setup in order to obtain identical DNA concentrations. After transfection cells were cultured in absence or presence of the calcineurin stimulators PMA and ionomycin (PMA/Io.) and the NFAT-pathway inhibitor Cyclosporin A (CspA). ** P

    Techniques Used: Expressing, In Vitro, Plasmid Preparation, Transfection, Cell Culture

    Validation of SARS-CoV Nsp1 interaction with immunophilins (cyclophilins PPIA, PPIB, PPIG, PPIH and FK506-binding protein FKBP1A) and calcipressin (RCAN3) by modified Lumier assay. Three versions of Nsp1 (Nsp1fl = aa 1–180, Nsp1N-terminus = aa 1–93 and Nsp1 C-terminus = aa119–180) and human cDNAs were cloned into protein A and Renilla Luciferase fusion vectors. Renilla -Nsp1 ( A ) or protein A-Nsp1 ( B ) was cotransfected with each respective cDNA into HEK293 cells. Complexes were purified via IgG-coated magnetic beads and Luciferase activity was determined as a measure for binding activity. As a positive control the very strongly interacting jun and fos genes were used. On the y-axis normalized signal to background ratios are shown.
    Figure Legend Snippet: Validation of SARS-CoV Nsp1 interaction with immunophilins (cyclophilins PPIA, PPIB, PPIG, PPIH and FK506-binding protein FKBP1A) and calcipressin (RCAN3) by modified Lumier assay. Three versions of Nsp1 (Nsp1fl = aa 1–180, Nsp1N-terminus = aa 1–93 and Nsp1 C-terminus = aa119–180) and human cDNAs were cloned into protein A and Renilla Luciferase fusion vectors. Renilla -Nsp1 ( A ) or protein A-Nsp1 ( B ) was cotransfected with each respective cDNA into HEK293 cells. Complexes were purified via IgG-coated magnetic beads and Luciferase activity was determined as a measure for binding activity. As a positive control the very strongly interacting jun and fos genes were used. On the y-axis normalized signal to background ratios are shown.

    Techniques Used: Binding Assay, Modification, Clone Assay, Luciferase, Purification, Magnetic Beads, Activity Assay, Positive Control

    Influence of Nsp1 on Interleukin promoters. HEK293 cells were transiently cotransfected with interleukin reporter plasmids IL2 luc ( A ), IL4 luc ( B ), IL8 luc ( C ) and expression plasmids encoding NFAT3, CnA and either SARS-CoV Nsp1fl or the empty plasmid vector. All experiments were also done with an additional overexpression of the Calcipressin RCAN3. After transfection cells were cultured in absence or with the calcineurin stimulators PMA/Io. and the inhibitor CspA. * P
    Figure Legend Snippet: Influence of Nsp1 on Interleukin promoters. HEK293 cells were transiently cotransfected with interleukin reporter plasmids IL2 luc ( A ), IL4 luc ( B ), IL8 luc ( C ) and expression plasmids encoding NFAT3, CnA and either SARS-CoV Nsp1fl or the empty plasmid vector. All experiments were also done with an additional overexpression of the Calcipressin RCAN3. After transfection cells were cultured in absence or with the calcineurin stimulators PMA/Io. and the inhibitor CspA. * P

    Techniques Used: Expressing, Plasmid Preparation, Over Expression, Transfection, Cell Culture

    25) Product Images from "Sterile α Motif Domain Containing 9 Is a Novel Cellular Interacting Partner to Low-Risk Type Human Papillomavirus E6 Proteins"

    Article Title: Sterile α Motif Domain Containing 9 Is a Novel Cellular Interacting Partner to Low-Risk Type Human Papillomavirus E6 Proteins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0149859

    HPV-11 E6 protein interacts with SAMD9 in HEK293 cells. (A) Input and co-immunoprecipitation of HA tagged E6 proteins in HEK293 cells that transiently express HA tagged GFP, 6E6, 11E6 and 16E6 proteins. Western blots were used to detect SAMD9 and HA in whole cell lysates and co-immunoprecipitated complexes using these cell lines. (B) The association between SAMD9 and E6 proteins was quantified and the density ratio of co-immunoprecipitated SAMD9 normalized by Immunoprecipitated HA was calculated. **** represents p
    Figure Legend Snippet: HPV-11 E6 protein interacts with SAMD9 in HEK293 cells. (A) Input and co-immunoprecipitation of HA tagged E6 proteins in HEK293 cells that transiently express HA tagged GFP, 6E6, 11E6 and 16E6 proteins. Western blots were used to detect SAMD9 and HA in whole cell lysates and co-immunoprecipitated complexes using these cell lines. (B) The association between SAMD9 and E6 proteins was quantified and the density ratio of co-immunoprecipitated SAMD9 normalized by Immunoprecipitated HA was calculated. **** represents p

    Techniques Used: Immunoprecipitation, Western Blot

    26) Product Images from "Functional Characterization of Schizophrenia-Associated Variation in CACNA1C"

    Article Title: Functional Characterization of Schizophrenia-Associated Variation in CACNA1C

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0157086

    EMSA for rs4765905 with HEK293 and SK-N-SH nuclear extracts. Nuclear extracts plus buffer are run in lanes 1 and 2. Probes plus buffer are run in lanes 3–5. Probes are incubated with nuclear extract as indicated by the “+” above the lane number in lanes 6–11. Lane 12 is buffer alone. Control allele is a positive control for the assay from an unrelated variant.
    Figure Legend Snippet: EMSA for rs4765905 with HEK293 and SK-N-SH nuclear extracts. Nuclear extracts plus buffer are run in lanes 1 and 2. Probes plus buffer are run in lanes 3–5. Probes are incubated with nuclear extract as indicated by the “+” above the lane number in lanes 6–11. Lane 12 is buffer alone. Control allele is a positive control for the assay from an unrelated variant.

    Techniques Used: Incubation, Positive Control, Variant Assay

    27) Product Images from "Mouse superkiller‐2‐like helicase DDX60 is dispensable for type I IFN induction and immunity to multiple viruses"

    Article Title: Mouse superkiller‐2‐like helicase DDX60 is dispensable for type I IFN induction and immunity to multiple viruses

    Journal: European Journal of Immunology

    doi: 10.1002/eji.201545794

    Overexpression of DDX60 or DDX60L does not induce type I IFNs. (A) Different human DDX60 and DDX60L constructs labeled A to H used in (B) for Western blot analysis and (C) IFN‐β promoter reporter assay. For (B), HEK293 cells were transfected with indicated plasmids and total cells lysates analyzed by Western blot. Membranes were probed with indicated antibodies. MYC‐hRIG‐I transfection was used as a control. In (C) HEK293 cells were cotransfected with an IFN‐β promoter firefly luciferase reporter, renilla luciferase control, and indicated amounts of hDDX60 and/or hDDX60L expression plasmids. Twenty‐four hours later, firefly luciferase (F‐luc) activity was measured and normalized to renilla luciferase (R‐luc) activity and to the water control. Plasmids coding 3xFlag‐hMAVS and 3xFlag‐hRIG‐I were used as positive controls, whereas LacZ was used as a negative one. (D) HEK293 cells were cotransfected with an ISRE promoter firefly luciferase reporter, a Renilla luciferase control, 200 ng/mL of control LacZ or 3xFlag‐hDDX60 expressing plasmid as well as indicated hRIG‐I, hMDA5, hTBK1, or hIRF‐3 expressing plasmids or treated with IFN‐A/D (1000 IU/mL). Twenty‐four hours later, luciferase activity was measured. (E) HEK293 cells were cotransfected with an IFN‐β promoter firefly luciferase reporter, a Renilla luciferase control, and 200 ng/mL of control LacZ or 3xFlag‐hDDX60 expressing plasmid. Twenty‐four hours later, cells were transfected with IVT‐RNA or poly(I:C) or infected with SeV. After overnight culture, luciferase activity was measured. For all experiments, one representative of three independent experimental repeats is shown.
    Figure Legend Snippet: Overexpression of DDX60 or DDX60L does not induce type I IFNs. (A) Different human DDX60 and DDX60L constructs labeled A to H used in (B) for Western blot analysis and (C) IFN‐β promoter reporter assay. For (B), HEK293 cells were transfected with indicated plasmids and total cells lysates analyzed by Western blot. Membranes were probed with indicated antibodies. MYC‐hRIG‐I transfection was used as a control. In (C) HEK293 cells were cotransfected with an IFN‐β promoter firefly luciferase reporter, renilla luciferase control, and indicated amounts of hDDX60 and/or hDDX60L expression plasmids. Twenty‐four hours later, firefly luciferase (F‐luc) activity was measured and normalized to renilla luciferase (R‐luc) activity and to the water control. Plasmids coding 3xFlag‐hMAVS and 3xFlag‐hRIG‐I were used as positive controls, whereas LacZ was used as a negative one. (D) HEK293 cells were cotransfected with an ISRE promoter firefly luciferase reporter, a Renilla luciferase control, 200 ng/mL of control LacZ or 3xFlag‐hDDX60 expressing plasmid as well as indicated hRIG‐I, hMDA5, hTBK1, or hIRF‐3 expressing plasmids or treated with IFN‐A/D (1000 IU/mL). Twenty‐four hours later, luciferase activity was measured. (E) HEK293 cells were cotransfected with an IFN‐β promoter firefly luciferase reporter, a Renilla luciferase control, and 200 ng/mL of control LacZ or 3xFlag‐hDDX60 expressing plasmid. Twenty‐four hours later, cells were transfected with IVT‐RNA or poly(I:C) or infected with SeV. After overnight culture, luciferase activity was measured. For all experiments, one representative of three independent experimental repeats is shown.

    Techniques Used: Over Expression, Construct, Labeling, Western Blot, Reporter Assay, Transfection, Luciferase, Expressing, Activity Assay, Plasmid Preparation, Infection

    DDX60 and DDX60L are IFN inducible Ski2‐like DExH helicases. (A) Schematic representation of the human Ski2‐like helicases DDX60 and DDX60L highlighting the helicase domain. Hel1 and Hel2 denote the consensus subdomains of the helicase domain. (B) Phylogenetic relationship of the Hel1 helicase subdomain (containing the DExH/D Walker B motif) of RIG‐I‐like and Ski2‐like helicases from human (hs), mouse (mm), fly ( Drosophila melanogaster , dm), yeast ( Saccharomyces cerevisae , sc), and nematode ( Caenorhabditis elegans , ce). (C) Microscopy image of live HeLa cells transfected with GFP‐tagged hDDX60. (D) Schematic representation of consensus ISRE binding sites found by computational analysis in the promoter of human and mouse DDX60 and in human DDX60L . (E) Murine NIH3T3 (left panel) and human HEK293 cells (right panel) were treated or not with recombinant IFN (+IFN‐A/D) for 8 h. The relative expression (RE) of human and murine DDX60 and human DDX60L was assessed by Q‐PCR and normalized to GAPDH. (F) RE of murine Ddx60 and Ddx58 ( Rig‐i ) from indicated murine organs. For PCR data, the mean (±SD) of triplicate technical replicates is shown. For experiments (C–E) one representative of three independent experimental repeats is shown, for (F) one of two is shown.
    Figure Legend Snippet: DDX60 and DDX60L are IFN inducible Ski2‐like DExH helicases. (A) Schematic representation of the human Ski2‐like helicases DDX60 and DDX60L highlighting the helicase domain. Hel1 and Hel2 denote the consensus subdomains of the helicase domain. (B) Phylogenetic relationship of the Hel1 helicase subdomain (containing the DExH/D Walker B motif) of RIG‐I‐like and Ski2‐like helicases from human (hs), mouse (mm), fly ( Drosophila melanogaster , dm), yeast ( Saccharomyces cerevisae , sc), and nematode ( Caenorhabditis elegans , ce). (C) Microscopy image of live HeLa cells transfected with GFP‐tagged hDDX60. (D) Schematic representation of consensus ISRE binding sites found by computational analysis in the promoter of human and mouse DDX60 and in human DDX60L . (E) Murine NIH3T3 (left panel) and human HEK293 cells (right panel) were treated or not with recombinant IFN (+IFN‐A/D) for 8 h. The relative expression (RE) of human and murine DDX60 and human DDX60L was assessed by Q‐PCR and normalized to GAPDH. (F) RE of murine Ddx60 and Ddx58 ( Rig‐i ) from indicated murine organs. For PCR data, the mean (±SD) of triplicate technical replicates is shown. For experiments (C–E) one representative of three independent experimental repeats is shown, for (F) one of two is shown.

    Techniques Used: Microscopy, Transfection, Binding Assay, Recombinant, Expressing, Polymerase Chain Reaction

    DDX60 does not precipitate with RIG‐I or MDA5. Western blot analyses were conducted on the following immunoprecipitates using indicated antibodies: (A) Flag precipitates from total cell lysates of HEK293T cells cotransfected for 24 h with GFP‐hDDX60 and 3xFlag‐tagged hMAVS, hRIG‐I, or hMDA5. (B) Flag precipitate from a total cell lysate of HEK293 cells stably expressing Flag‐hDDX60 transfected for 24 h with a MYC‐hRIG‐I expression plasmid. (C) HA precipitates from total cell lysates of HEK293T cells cotransfected for 48 h with 3xHA‐hDDX60 and 3xFlag‐tagged hDDX60 or hRIG‐I. (D) Flag and control IgG precipitates from total cell lysates of HEK293 cells stably expressing Flag‐hRIG‐I transfected with poly(I:C) or poly(dA:dT) or infected with SeV (MOI 1, 16 h). Total cell lysate from HEK293 cells stably expressing Flag‐hDDX60 were also included as a control. In (A–D) input samples (10% of total lysates before IP) were also analyzed. For experiments (A–C) one representative of three independent experimental repeats is shown, for (D) one of two is shown.
    Figure Legend Snippet: DDX60 does not precipitate with RIG‐I or MDA5. Western blot analyses were conducted on the following immunoprecipitates using indicated antibodies: (A) Flag precipitates from total cell lysates of HEK293T cells cotransfected for 24 h with GFP‐hDDX60 and 3xFlag‐tagged hMAVS, hRIG‐I, or hMDA5. (B) Flag precipitate from a total cell lysate of HEK293 cells stably expressing Flag‐hDDX60 transfected for 24 h with a MYC‐hRIG‐I expression plasmid. (C) HA precipitates from total cell lysates of HEK293T cells cotransfected for 48 h with 3xHA‐hDDX60 and 3xFlag‐tagged hDDX60 or hRIG‐I. (D) Flag and control IgG precipitates from total cell lysates of HEK293 cells stably expressing Flag‐hRIG‐I transfected with poly(I:C) or poly(dA:dT) or infected with SeV (MOI 1, 16 h). Total cell lysate from HEK293 cells stably expressing Flag‐hDDX60 were also included as a control. In (A–D) input samples (10% of total lysates before IP) were also analyzed. For experiments (A–C) one representative of three independent experimental repeats is shown, for (D) one of two is shown.

    Techniques Used: Western Blot, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Infection

    IP of DDX60 does not coprecipitate stimulatory RNA. (A) Diagram of experiment conducted in (B) where the stimulatory capacity of nucleic acid purified from stable HEK293‐Flag‐hDDX60 cells infected with SeV or PR8ΔNS1 (MOI 1) was assessed by IFN‐β promoter reporter assay. Water was used as a control. (C) Diagram of experiment conducted in (D, E, and F) where Flag‐hDDX60‐stable HEK293 cells were transfected with (D) IVT‐RNA or (E) poly(dA:dT) or infected with (F) SeV. Sixteen hours later, Flag‐hDDX60 was immunoprecipitated with an anti‐Flag antibody. An isotype‐matched IgG IP was included as control. RNA was then purified from precipitates and stimulatory capacity was assessed by IFN‐β promoter reporter assay (above). IP samples were also tested for Flag expression by Western blot (below). Experiment was conducted in conjunction with HEK293‐Flag‐hRIG‐I cells. (G) Diagram of experiment conducted in (H) where HEK293T cells were transfected with Flag‐hDDX60. Forty‐eight hours later, Flag‐hDDX60 was immunoprecipitated with an anti‐Flag antibody and an isotype‐matched IgG IP was included as control. Flag‐hDDX60‐coated IP beads as well as control beads were incubated with RNA isolated from IAV (IAV vRNA). Following extensive washes, RNA was purified from precipitates and stimulatory capacity was assessed by IFN‐β promoter reporter assay. IP samples were tested for Flag expression by Western blot. The experiment was conducted in conjunction with Flag‐hRIG‐I transfection. For all experiments one representative of three independent experimental repeats is shown.
    Figure Legend Snippet: IP of DDX60 does not coprecipitate stimulatory RNA. (A) Diagram of experiment conducted in (B) where the stimulatory capacity of nucleic acid purified from stable HEK293‐Flag‐hDDX60 cells infected with SeV or PR8ΔNS1 (MOI 1) was assessed by IFN‐β promoter reporter assay. Water was used as a control. (C) Diagram of experiment conducted in (D, E, and F) where Flag‐hDDX60‐stable HEK293 cells were transfected with (D) IVT‐RNA or (E) poly(dA:dT) or infected with (F) SeV. Sixteen hours later, Flag‐hDDX60 was immunoprecipitated with an anti‐Flag antibody. An isotype‐matched IgG IP was included as control. RNA was then purified from precipitates and stimulatory capacity was assessed by IFN‐β promoter reporter assay (above). IP samples were also tested for Flag expression by Western blot (below). Experiment was conducted in conjunction with HEK293‐Flag‐hRIG‐I cells. (G) Diagram of experiment conducted in (H) where HEK293T cells were transfected with Flag‐hDDX60. Forty‐eight hours later, Flag‐hDDX60 was immunoprecipitated with an anti‐Flag antibody and an isotype‐matched IgG IP was included as control. Flag‐hDDX60‐coated IP beads as well as control beads were incubated with RNA isolated from IAV (IAV vRNA). Following extensive washes, RNA was purified from precipitates and stimulatory capacity was assessed by IFN‐β promoter reporter assay. IP samples were tested for Flag expression by Western blot. The experiment was conducted in conjunction with Flag‐hRIG‐I transfection. For all experiments one representative of three independent experimental repeats is shown.

    Techniques Used: Purification, Infection, Reporter Assay, Transfection, Immunoprecipitation, Expressing, Western Blot, Incubation, Isolation

    28) Product Images from "A Becn1 mutation mediates hyperactive autophagic sequestration of amyloid oligomers and improved cognition in Alzheimer's disease"

    Article Title: A Becn1 mutation mediates hyperactive autophagic sequestration of amyloid oligomers and improved cognition in Alzheimer's disease

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1006962

    An autophagy-inducing compound ML246 reduces amyloid load in an autophagy-dependent manner in vitro and in vivo. (A) Chemical structure of ML246. (B) Dot-blot assays (left) and quantification (right) of secreted Aβ42 levels in conditioned media of HEK293 cells stably expressing APP treated with vehicle (DMSO) or ML246 for 24 h, immunostained with anti-Aβ42 antibody. Cells were transfected with non-targeting control (NC) or ATG7 siRNA 24 h prior to ML246 treatment. Results are quantified from 4 independent experiments. (C) Representative images (left) and quantification (right) of TUNEL signals (red) in WT primary cortical neurons treated with conditioned media from ( B ) for 24 h. Nuclei were stained with DAPI. Scale bar, 100 μm. N = 10 fields (each field containing 20–30 neurons). (D, E) Representative images (upper) and quantification (lower) of dot-blot assays on soluble (D) and insoluble (E) Aβ42 levels in brain samples of 6-month old 5XFAD and 5XFAD Becn1 +/- KO mice after 5 weeks of ML246 treatment, immunostained with anti-Aβ42 antibody. Total protein loading was labeled by Ponceau S. Triplicate experiments from 4–5 mice in each group were shown. Results represent mean ± s.e.m. NS, not significant; *, P
    Figure Legend Snippet: An autophagy-inducing compound ML246 reduces amyloid load in an autophagy-dependent manner in vitro and in vivo. (A) Chemical structure of ML246. (B) Dot-blot assays (left) and quantification (right) of secreted Aβ42 levels in conditioned media of HEK293 cells stably expressing APP treated with vehicle (DMSO) or ML246 for 24 h, immunostained with anti-Aβ42 antibody. Cells were transfected with non-targeting control (NC) or ATG7 siRNA 24 h prior to ML246 treatment. Results are quantified from 4 independent experiments. (C) Representative images (left) and quantification (right) of TUNEL signals (red) in WT primary cortical neurons treated with conditioned media from ( B ) for 24 h. Nuclei were stained with DAPI. Scale bar, 100 μm. N = 10 fields (each field containing 20–30 neurons). (D, E) Representative images (upper) and quantification (lower) of dot-blot assays on soluble (D) and insoluble (E) Aβ42 levels in brain samples of 6-month old 5XFAD and 5XFAD Becn1 +/- KO mice after 5 weeks of ML246 treatment, immunostained with anti-Aβ42 antibody. Total protein loading was labeled by Ponceau S. Triplicate experiments from 4–5 mice in each group were shown. Results represent mean ± s.e.m. NS, not significant; *, P

    Techniques Used: In Vitro, In Vivo, Dot Blot, Stable Transfection, Expressing, Transfection, TUNEL Assay, Staining, Mouse Assay, Labeling

    29) Product Images from "Gelofusine Attenuates Tubulointerstitial Injury Induced by cRGD-Conjugated siRNA by Regulating the TLR3 Signaling Pathway"

    Article Title: Gelofusine Attenuates Tubulointerstitial Injury Induced by cRGD-Conjugated siRNA by Regulating the TLR3 Signaling Pathway

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2018.03.006

    Influence and Mechanism of Action of Gelofusine on Immunogenicity and Apoptosis Induced by cRGD-siRNA In Vitro (A) The cellular uptake levels and distribution of cRGD-siRNA in HK-2 cells. The cells were transfected with different concentrations of cRGD-siNC-Cy5. For the Gelofusine group, before transfection with cRGD-siNC-Cy5, the cells were pretreated with 100 μg/mL Gelofusine. After 24-hr treatment, the cells were collected and measured by flow cytometry or fixed and visualized using confocal laser microscopy. Cell nuclei were counterstained with DAPI (blue), and siRNA was labeled with Cy5 (red). (B) Analysis of TLR3 expression on the surface and total area of HK-2 and HEK293 cells. The cells were collected and measured by flow cytometry. (C) Analysis of apoptosis of HK-2 cells using different treatments. HK-2 cells were transfected with different concentrations of cRGD-siNC. For the Gelofusine group, before transfection with cRGD-siNC, the cells were pretreated with 100 μg/mL Gelofusine. After 48-hr transfection, the cells were collected and analyzed by flow cytometry with Annexin V and propidium iodide. (D and E) Quantitative analysis of IL-6 (D) and IFN-β (E) levels using ELISA. After 24-hr treatment, the cell supernatant was collected and analyzed using ELISA. (F) The distribution of p65/RelA in HK-2 cells when using different treatments. After 24-hr treatment, the cells were fixed and visualized using confocal laser microscopy. Cell nuclei were counterstained with DAPI (blue), and p65/RelA was detected with an antibody conjugated with Alexa Fluor 488 (green). (G and H). Effects on the NF-κB pathway (G) and cleaved caspase-3 expression (H) using different treatments. After 48-hr treatment, the cells were collected for western blot analysis. The expression of protein was calculated compared with the expression of β-actin (ACTB). *p
    Figure Legend Snippet: Influence and Mechanism of Action of Gelofusine on Immunogenicity and Apoptosis Induced by cRGD-siRNA In Vitro (A) The cellular uptake levels and distribution of cRGD-siRNA in HK-2 cells. The cells were transfected with different concentrations of cRGD-siNC-Cy5. For the Gelofusine group, before transfection with cRGD-siNC-Cy5, the cells were pretreated with 100 μg/mL Gelofusine. After 24-hr treatment, the cells were collected and measured by flow cytometry or fixed and visualized using confocal laser microscopy. Cell nuclei were counterstained with DAPI (blue), and siRNA was labeled with Cy5 (red). (B) Analysis of TLR3 expression on the surface and total area of HK-2 and HEK293 cells. The cells were collected and measured by flow cytometry. (C) Analysis of apoptosis of HK-2 cells using different treatments. HK-2 cells were transfected with different concentrations of cRGD-siNC. For the Gelofusine group, before transfection with cRGD-siNC, the cells were pretreated with 100 μg/mL Gelofusine. After 48-hr transfection, the cells were collected and analyzed by flow cytometry with Annexin V and propidium iodide. (D and E) Quantitative analysis of IL-6 (D) and IFN-β (E) levels using ELISA. After 24-hr treatment, the cell supernatant was collected and analyzed using ELISA. (F) The distribution of p65/RelA in HK-2 cells when using different treatments. After 24-hr treatment, the cells were fixed and visualized using confocal laser microscopy. Cell nuclei were counterstained with DAPI (blue), and p65/RelA was detected with an antibody conjugated with Alexa Fluor 488 (green). (G and H). Effects on the NF-κB pathway (G) and cleaved caspase-3 expression (H) using different treatments. After 48-hr treatment, the cells were collected for western blot analysis. The expression of protein was calculated compared with the expression of β-actin (ACTB). *p

    Techniques Used: In Vitro, Transfection, Flow Cytometry, Cytometry, Microscopy, Labeling, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

    30) Product Images from "Inverted repeat Alu elements in the human lincRNA-p21 adopt a conserved secondary structure that regulates RNA function"

    Article Title: Inverted repeat Alu elements in the human lincRNA-p21 adopt a conserved secondary structure that regulates RNA function

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw599

    The human lincRNA-21 is a single exon lncRNA that contains IR Alu elements. ( A ) Diagram of precursor (Pre-RNA) and mature mouse lincRNA-p21 (mLincRNA-p21) and of the previously reported partial sequence of human lincRNA-p21 (hLincRNA-p21) [adapted from Yoon et al . ( 9 )]. The asterisk represents the p53 protein binding site. The shaded box represents a region of sequence homology between mLincRNA-p21 and hLincRNA-p21. ( B ) RT-PCR performed on total RNA from HEK293, Hela, H1299, MCF-10A, HCT-116 and MCF-7 cell lines and on primary dermal fibroblasts. Four different primer pairs were used to amplify portions of exon 1 (E1), the putative exon1-intron junction (US), the putative exon1–exon2 junction (Sp) and exon 2 (E2). H stands for housekeeping gene (tRNA-Lys for HeLa and ACTB for all other cell lines). MW is a DNA size ladder expressed in base pairs. ( C ) Comparative diagram of the full-length sequence isoforms of hLincRNA-p21 as obtained by 3′ RACE on HEK293 cells and the previously reported partial sequence of hLincRNA-p21. ( D ) Schematic representation of hLincRNA-p21 with associated UCSC Genome Browser tracks depicting ENCODE H3K4me3 and H3K27Ac ChIP-seq coverage as well as CSHL Long RNA-seq coverage ( 26 ). ( E ) Northern blot analysis of hLincRNA-p21 in nuclear RNA from HEK293 and HCT-116 cell lines and of total and nuclear RNA from primary dermal fibroblasts. A probe hybridizing with the 5′ end of hLincRNA-p21 was used. ( F ) Maximum CSF scores of hLincRNA-p21 isoforms and CDKN1A (p21) RNAs determined by analysis with PhyloCSF ( 27 ). ( G ) Quantification of hLincRNA-p21 copy number per cell determined in the HCT-116 cell line by qRT-PCR.
    Figure Legend Snippet: The human lincRNA-21 is a single exon lncRNA that contains IR Alu elements. ( A ) Diagram of precursor (Pre-RNA) and mature mouse lincRNA-p21 (mLincRNA-p21) and of the previously reported partial sequence of human lincRNA-p21 (hLincRNA-p21) [adapted from Yoon et al . ( 9 )]. The asterisk represents the p53 protein binding site. The shaded box represents a region of sequence homology between mLincRNA-p21 and hLincRNA-p21. ( B ) RT-PCR performed on total RNA from HEK293, Hela, H1299, MCF-10A, HCT-116 and MCF-7 cell lines and on primary dermal fibroblasts. Four different primer pairs were used to amplify portions of exon 1 (E1), the putative exon1-intron junction (US), the putative exon1–exon2 junction (Sp) and exon 2 (E2). H stands for housekeeping gene (tRNA-Lys for HeLa and ACTB for all other cell lines). MW is a DNA size ladder expressed in base pairs. ( C ) Comparative diagram of the full-length sequence isoforms of hLincRNA-p21 as obtained by 3′ RACE on HEK293 cells and the previously reported partial sequence of hLincRNA-p21. ( D ) Schematic representation of hLincRNA-p21 with associated UCSC Genome Browser tracks depicting ENCODE H3K4me3 and H3K27Ac ChIP-seq coverage as well as CSHL Long RNA-seq coverage ( 26 ). ( E ) Northern blot analysis of hLincRNA-p21 in nuclear RNA from HEK293 and HCT-116 cell lines and of total and nuclear RNA from primary dermal fibroblasts. A probe hybridizing with the 5′ end of hLincRNA-p21 was used. ( F ) Maximum CSF scores of hLincRNA-p21 isoforms and CDKN1A (p21) RNAs determined by analysis with PhyloCSF ( 27 ). ( G ) Quantification of hLincRNA-p21 copy number per cell determined in the HCT-116 cell line by qRT-PCR.

    Techniques Used: Sequencing, Protein Binding, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, RNA Sequencing Assay, Northern Blot, Quantitative RT-PCR

    31) Product Images from "Membrane progesterone receptor beta (mPRβ/Paqr8) promotes progesterone-dependent neurite outgrowth in PC12 neuronal cells via non-G protein-coupled receptor (GPCR) signaling"

    Article Title: Membrane progesterone receptor beta (mPRβ/Paqr8) promotes progesterone-dependent neurite outgrowth in PC12 neuronal cells via non-G protein-coupled receptor (GPCR) signaling

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-05423-9

    mPRβ stimulation by progesterone promotes ERK phosphorylation via non-GPCR signaling. ( a ) Prediction of transmembrane regions of mPRβ by using TMHMM 2.0 program. ( b ) Localization of mPRβ or GPR41 with epitope tags at either end. ( c ) The expression of mPRβ from the Flp-In locus was induced by treatment with 10 μg/mL doxycycline. After 24 h in culture, Flp in mPRβ T-Rex HEK293 cells were examined by immunochemistry with an anti-E-tag antibody. Green signals indicate mPRβ expression and blue signals indicate cell nuclei counter-stained with DAPI. (Scale bar = 20 μm). ( d ) Expression of mPRβ mRNA in Flp in mPRβ T-Rex HEK293 cells. Expression of mPRβ was measured using quantitative RT-PCR. 18S mRNA expression was used as an internal control. (n = 3). ( e ) Mobilization of [Ca 2+ ] i induced by progesterone was monitored in Flp in mPRβ T-Rex HEK293 cells, and data are presented as relative Ca 2+ intensity. After 2 h in culture, cells were treated with or without 10 μg/mL doxycycline. (n = 3). ( f ) cAMP levels in response to progesterone treatment in Flp in mPRβ T-Rex HEK293 cells. After 24 h in culture, cells were treated with or without 10 μg/mL doxycycline and further cultured for 24 h. Cells pre-cultured with IBMX for 30 min were cultured in the presence of progesterone for 10 min. The cAMP levels in the cells were determined by using a cAMP EIA kit. (n = 4). ( g ) Effects of progesterone on AMPK phosphorylation in Flp in mPRβ T-Rex HEK293 cells. After 24 h in culture with or without doxycycline (10 μg/mL), cells were further cultured for 24 h in serum-free DMEM. The cells were cultured in the presence of progesterone for 10 min. (n = 5) ( h ) Effects of progesterone on ERK1/2 phosphorylation in Flp in mPRβ T-Rex HEK293 cells. After 24 h in culture with or without doxycycline (10 μg/mL), cells were further cultured for 24 h in serum-free DMEM. The cells were cultured in the presence of progesterone for 10 min. Dox: Doxycycline. (n = 3).
    Figure Legend Snippet: mPRβ stimulation by progesterone promotes ERK phosphorylation via non-GPCR signaling. ( a ) Prediction of transmembrane regions of mPRβ by using TMHMM 2.0 program. ( b ) Localization of mPRβ or GPR41 with epitope tags at either end. ( c ) The expression of mPRβ from the Flp-In locus was induced by treatment with 10 μg/mL doxycycline. After 24 h in culture, Flp in mPRβ T-Rex HEK293 cells were examined by immunochemistry with an anti-E-tag antibody. Green signals indicate mPRβ expression and blue signals indicate cell nuclei counter-stained with DAPI. (Scale bar = 20 μm). ( d ) Expression of mPRβ mRNA in Flp in mPRβ T-Rex HEK293 cells. Expression of mPRβ was measured using quantitative RT-PCR. 18S mRNA expression was used as an internal control. (n = 3). ( e ) Mobilization of [Ca 2+ ] i induced by progesterone was monitored in Flp in mPRβ T-Rex HEK293 cells, and data are presented as relative Ca 2+ intensity. After 2 h in culture, cells were treated with or without 10 μg/mL doxycycline. (n = 3). ( f ) cAMP levels in response to progesterone treatment in Flp in mPRβ T-Rex HEK293 cells. After 24 h in culture, cells were treated with or without 10 μg/mL doxycycline and further cultured for 24 h. Cells pre-cultured with IBMX for 30 min were cultured in the presence of progesterone for 10 min. The cAMP levels in the cells were determined by using a cAMP EIA kit. (n = 4). ( g ) Effects of progesterone on AMPK phosphorylation in Flp in mPRβ T-Rex HEK293 cells. After 24 h in culture with or without doxycycline (10 μg/mL), cells were further cultured for 24 h in serum-free DMEM. The cells were cultured in the presence of progesterone for 10 min. (n = 5) ( h ) Effects of progesterone on ERK1/2 phosphorylation in Flp in mPRβ T-Rex HEK293 cells. After 24 h in culture with or without doxycycline (10 μg/mL), cells were further cultured for 24 h in serum-free DMEM. The cells were cultured in the presence of progesterone for 10 min. Dox: Doxycycline. (n = 3).

    Techniques Used: Expressing, Staining, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay

    32) Product Images from "Protein instability, haploinsufficiency, and cortical hyper-excitability underlie STXBP1 encephalopathy"

    Article Title: Protein instability, haploinsufficiency, and cortical hyper-excitability underlie STXBP1 encephalopathy

    Journal: Brain

    doi: 10.1093/brain/awy046

    Cellular stability of wild-type and human disease variants of Munc18-1 in HEK293 cells and neurons. ( A ) Immunochemistry of HEK293 cells infected with wild-type Munc18-1 (WT), C180Y, M443R, C522R and T574P constructs stained for Munc18-1, EGFP and Golgi marker (GM130). ( B ) Normalized Munc18-1 levels in HEK293 cells after viral infection with wild-type, C180Y, M443R, C522R and T574P constructs. The inset shows representative western blot of HEK293 cells after viral infection; n = 5, 5, 5, 2 and 2, respectively. ( C ) Western blot analysis of Munc18-1 protein levels 0, 6, 12, 24 and 30 h after block of protein synthesis with cycloheximide for HEK293 cells infected with wild-type, C180Y, M433R, C522R or T574P constructs. The infection with wild-type construct was used as a control for all performed western blot analysis. ( D ) Quantitative analysis of the Munc18-1 protein expression from western blots in HEK cells represented in C . ( E ) Western blot analysis of Munc18-1 protein levels 0, 12, 24 and 36 h after block of protein synthesis with cycloheximide for wild-type, C180Y, M433R, C522R or T574P constructs in Stxbp1 null neurons. The infection with wild-type construct was used as a control for all performed western blot analysis. ( F ) Quantitative analysis of the Munc18-1 protein expression from western blots in neurons represe nted in E . ( G–I ) Normalized Munc18-1 protein levels from three constructs expressed in HEK cells treated with MG132, Leupeptin or Pepstatin; n = 3, 2 and 2, respectively.
    Figure Legend Snippet: Cellular stability of wild-type and human disease variants of Munc18-1 in HEK293 cells and neurons. ( A ) Immunochemistry of HEK293 cells infected with wild-type Munc18-1 (WT), C180Y, M443R, C522R and T574P constructs stained for Munc18-1, EGFP and Golgi marker (GM130). ( B ) Normalized Munc18-1 levels in HEK293 cells after viral infection with wild-type, C180Y, M443R, C522R and T574P constructs. The inset shows representative western blot of HEK293 cells after viral infection; n = 5, 5, 5, 2 and 2, respectively. ( C ) Western blot analysis of Munc18-1 protein levels 0, 6, 12, 24 and 30 h after block of protein synthesis with cycloheximide for HEK293 cells infected with wild-type, C180Y, M433R, C522R or T574P constructs. The infection with wild-type construct was used as a control for all performed western blot analysis. ( D ) Quantitative analysis of the Munc18-1 protein expression from western blots in HEK cells represented in C . ( E ) Western blot analysis of Munc18-1 protein levels 0, 12, 24 and 36 h after block of protein synthesis with cycloheximide for wild-type, C180Y, M433R, C522R or T574P constructs in Stxbp1 null neurons. The infection with wild-type construct was used as a control for all performed western blot analysis. ( F ) Quantitative analysis of the Munc18-1 protein expression from western blots in neurons represe nted in E . ( G–I ) Normalized Munc18-1 protein levels from three constructs expressed in HEK cells treated with MG132, Leupeptin or Pepstatin; n = 3, 2 and 2, respectively.

    Techniques Used: Infection, Construct, Staining, Marker, Western Blot, Blocking Assay, Expressing

    33) Product Images from "Experimental autoimmune encephalomyelitis (EAE) up-regulates the mitochondrial activity and manganese superoxide dismutase (MnSOD) in the mouse renal cortex"

    Article Title: Experimental autoimmune encephalomyelitis (EAE) up-regulates the mitochondrial activity and manganese superoxide dismutase (MnSOD) in the mouse renal cortex

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0196277

    Monensin elevated the mitochondrial MnSOD protein level without a significant effect on the cytosolic MnSOD protein abundance (A, n = 5). Ouabain (4 nM) inhibited the effect of monensin on the mitochondrial MnSOD protein (B, n = 5). Membranes were probed with antibodies against aldose reductase (AR, cytosolic marker) and cytochrome c oxidase subunit IV (COX4, mitochondrial marker) to show adequate separation of these two compartments and with actin antibody to show roughly equal loading. Monensin had no significant effect on the total activity of cytosolic or mitochondrial MnSOD (C, n = 5), but significantly reduced the specific activity of mitochondrial MnSOD and this effect was blocked by ouabain (D, n = 5). HEK293 cells were treated as in Fig 5 . The total and specific MnSOD activities were measured as in Fig 4 , but the total and specific activities were normalized to the control in each experiment. (*p
    Figure Legend Snippet: Monensin elevated the mitochondrial MnSOD protein level without a significant effect on the cytosolic MnSOD protein abundance (A, n = 5). Ouabain (4 nM) inhibited the effect of monensin on the mitochondrial MnSOD protein (B, n = 5). Membranes were probed with antibodies against aldose reductase (AR, cytosolic marker) and cytochrome c oxidase subunit IV (COX4, mitochondrial marker) to show adequate separation of these two compartments and with actin antibody to show roughly equal loading. Monensin had no significant effect on the total activity of cytosolic or mitochondrial MnSOD (C, n = 5), but significantly reduced the specific activity of mitochondrial MnSOD and this effect was blocked by ouabain (D, n = 5). HEK293 cells were treated as in Fig 5 . The total and specific MnSOD activities were measured as in Fig 4 , but the total and specific activities were normalized to the control in each experiment. (*p

    Techniques Used: Marker, Activity Assay

    Catalase reduced the monensin-induced increase of mitochondrial MnSOD protein abundance (A, n = 5). The same membrane used in Fig 9A was incubated with the antibodies against MnSOD, AR, COX4 and actin (A). Catalase had no significant effect on the total MnSOD activity (B, n = 5), but reversed the monensin-induced inhibition of the specific MnSOD activity (C, n = 5). Knockdown of MnSOD reduced cellular ATP level with or without monensin (D, n = 4). The protein levels, total and specific activities of MnSOD were measured as in Fig 7 . HEK293 cells were transfected with 80 nM control or MnSOD siRNA overnight and treated with or without monensin for 24 hours. The cellular ATP levels were measured as in Fig 5 . (*p
    Figure Legend Snippet: Catalase reduced the monensin-induced increase of mitochondrial MnSOD protein abundance (A, n = 5). The same membrane used in Fig 9A was incubated with the antibodies against MnSOD, AR, COX4 and actin (A). Catalase had no significant effect on the total MnSOD activity (B, n = 5), but reversed the monensin-induced inhibition of the specific MnSOD activity (C, n = 5). Knockdown of MnSOD reduced cellular ATP level with or without monensin (D, n = 4). The protein levels, total and specific activities of MnSOD were measured as in Fig 7 . HEK293 cells were transfected with 80 nM control or MnSOD siRNA overnight and treated with or without monensin for 24 hours. The cellular ATP levels were measured as in Fig 5 . (*p

    Techniques Used: Incubation, Activity Assay, Inhibition, Transfection

    Monensin increased the mitochondrial ROS in the isolated mitochondria (A, n = 5) and in live HEK293 cells (B, n = 4 and C). Ouabain inhibited the effect of monensin. (A) HEK293 cells were treated as in Fig 5 and ROS from isolated mitochondria were measured as in Fig 2A . B is average of 4 independent flow cytometry analyses of mitochondrial ROS in live HEK293 cells. MFI: mean fluorescence intensity. C is a representative flow cytometry analysis. After HEK293 cells were incubated with 4 nM ouabain for 60 min, monensin (10 μM) was added into the medium and the cells were incubated for an additional 60 min, then Mito-sox Red (2.5 μM) was added and the cells were incubated for another 60 min before they were collected for analysis. (*p
    Figure Legend Snippet: Monensin increased the mitochondrial ROS in the isolated mitochondria (A, n = 5) and in live HEK293 cells (B, n = 4 and C). Ouabain inhibited the effect of monensin. (A) HEK293 cells were treated as in Fig 5 and ROS from isolated mitochondria were measured as in Fig 2A . B is average of 4 independent flow cytometry analyses of mitochondrial ROS in live HEK293 cells. MFI: mean fluorescence intensity. C is a representative flow cytometry analysis. After HEK293 cells were incubated with 4 nM ouabain for 60 min, monensin (10 μM) was added into the medium and the cells were incubated for an additional 60 min, then Mito-sox Red (2.5 μM) was added and the cells were incubated for another 60 min before they were collected for analysis. (*p

    Techniques Used: Isolation, Flow Cytometry, Cytometry, Fluorescence, Incubation

    Catalase reduced the monensin-induced increases of mitochondrial ROS in the isolated mitochondria (A) and in live HEK293 cells (B, n = 5 and C, n = 5). Mitochondrial ROS were measured as in Fig 6 except ouabain was replaced by catalase (400U/ml) (*p
    Figure Legend Snippet: Catalase reduced the monensin-induced increases of mitochondrial ROS in the isolated mitochondria (A) and in live HEK293 cells (B, n = 5 and C, n = 5). Mitochondrial ROS were measured as in Fig 6 except ouabain was replaced by catalase (400U/ml) (*p

    Techniques Used: Isolation

    Monensin (10 μM) increased the mitochondrial complex II activity (A, n = 5), but decreased the complex I activity (B, n = 5) and had no significant effect on the complex IV activity (C, n = 4). Ouabain (4 nM) blocked the effect of monensin on the complex II activity (A). Monensin had no significant effect on the cytosolic ATP level, but decreased the mitochondrial ATP level (D, n = 6). HEK293 cells were pre-incubated with ouabain for 60 min and then monensin was added for 24 hours before they were collected. The complex I, II and IV activities were measured as in Fig 1A and 1B . ATP level was measured by a luminescence-based ATP Determination Kit from Molecular Probes. (*p
    Figure Legend Snippet: Monensin (10 μM) increased the mitochondrial complex II activity (A, n = 5), but decreased the complex I activity (B, n = 5) and had no significant effect on the complex IV activity (C, n = 4). Ouabain (4 nM) blocked the effect of monensin on the complex II activity (A). Monensin had no significant effect on the cytosolic ATP level, but decreased the mitochondrial ATP level (D, n = 6). HEK293 cells were pre-incubated with ouabain for 60 min and then monensin was added for 24 hours before they were collected. The complex I, II and IV activities were measured as in Fig 1A and 1B . ATP level was measured by a luminescence-based ATP Determination Kit from Molecular Probes. (*p

    Techniques Used: Activity Assay, Incubation

    34) Product Images from "Identification and characterization of a novel folliculin-interacting protein FNIP2"

    Article Title: Identification and characterization of a novel folliculin-interacting protein FNIP2

    Journal:

    doi: 10.1016/j.gene.2008.02.022

    FNIP1 and FNIP2 multimer formation. (A) Doxycycline-inducible HA-FLCN-expressing HEK293 cells co-transfected with V5-FNIP1 and Flag-FNIP2 were immunoprecipitated with anti-V5 or anti-Flag antibody followed by western blotting with indicated antibodies.
    Figure Legend Snippet: FNIP1 and FNIP2 multimer formation. (A) Doxycycline-inducible HA-FLCN-expressing HEK293 cells co-transfected with V5-FNIP1 and Flag-FNIP2 were immunoprecipitated with anti-V5 or anti-Flag antibody followed by western blotting with indicated antibodies.

    Techniques Used: Expressing, Transfection, Immunoprecipitation, Western Blot

    35) Product Images from "Polycystin-1 regulates bone development through an interaction with the transcriptional coactivator TAZ"

    Article Title: Polycystin-1 regulates bone development through an interaction with the transcriptional coactivator TAZ

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddy322

    PC1-CTT binds to TAZ without disrupting the TAZ-14-3-3 interaction. (A) HEK293 cells were co-transfected with FLAG-TAZ or FLAG-YAP and HA-PC1-CTT. Cell lysates were subjected to immunoprecipitation using anti-FLAG sepharose, then blotted with the indicated antibodies. (B) HEK293 cells were co-transfected with FLAG-TAZ and HA-PC1-CTT. Cell lysates were subjected to immunoprecipitation using anti-HA sepharose, then blotted with the indicated antibodies. (C) A GST-tagged construct containing the C-terminal 91 amino acids of the PC1-CTT (p91) was produced in BL21 bacteria and purified on glutathione-sepharose 4B beads. The GST-p91 coated glutathione beads were then exposed to lysates from HEK293 cells expressing FLAG-TAZ and the resulting complexes were blotted with the indicated antibodies. (D) HEK293 cells were co-transfected with FLAG-TAZ(WT) or FLAG-TAZ(S89A) and HA-PC1-CTT. Cell lysates were subjected to immunoprecipitation using anti-FLAG sepharose, then blotted with the indicated antibodies.
    Figure Legend Snippet: PC1-CTT binds to TAZ without disrupting the TAZ-14-3-3 interaction. (A) HEK293 cells were co-transfected with FLAG-TAZ or FLAG-YAP and HA-PC1-CTT. Cell lysates were subjected to immunoprecipitation using anti-FLAG sepharose, then blotted with the indicated antibodies. (B) HEK293 cells were co-transfected with FLAG-TAZ and HA-PC1-CTT. Cell lysates were subjected to immunoprecipitation using anti-HA sepharose, then blotted with the indicated antibodies. (C) A GST-tagged construct containing the C-terminal 91 amino acids of the PC1-CTT (p91) was produced in BL21 bacteria and purified on glutathione-sepharose 4B beads. The GST-p91 coated glutathione beads were then exposed to lysates from HEK293 cells expressing FLAG-TAZ and the resulting complexes were blotted with the indicated antibodies. (D) HEK293 cells were co-transfected with FLAG-TAZ(WT) or FLAG-TAZ(S89A) and HA-PC1-CTT. Cell lysates were subjected to immunoprecipitation using anti-FLAG sepharose, then blotted with the indicated antibodies.

    Techniques Used: Transfection, Immunoprecipitation, Construct, Produced, Purification, Expressing

    PC1-CTT activation of RunX2 requires p300, and PC1-CTT enhances the TAZ-p300 association (A) HEK293 cells were transfected with either control shRNA (shControl) or shRNA directed against human p300 (shp300). mRNA knockdown efficiency was assessed by qRT-PCR. (B) HEK293 cells were transfected with shControl or shp300. After 48h, the cells were super-transfected with RunX2-luciferase and Renilla-luciferase alone, or in the presence of the PC1-CTT. Luciferase values were measured 24 h after RunX2-lucferase transfection (72 h total after initial shRNA treatment). (C) HEK293 cells were transfected with Myc-p300, FLAG-TAZ, and HA-PC1-CTT where indicated. Cell lysates were incubated with anti-FLAG beads, complexes were eluted in SDS-PAGE loading buffer and run on a 10% SDS- polyacrylamide gel and blotted with the indicated antibodies. Densitometry was performed on the immunoprecipitated p300 band using image analysis software (right panel). Results are expressed as mean ± SE from four independent experiments.
    Figure Legend Snippet: PC1-CTT activation of RunX2 requires p300, and PC1-CTT enhances the TAZ-p300 association (A) HEK293 cells were transfected with either control shRNA (shControl) or shRNA directed against human p300 (shp300). mRNA knockdown efficiency was assessed by qRT-PCR. (B) HEK293 cells were transfected with shControl or shp300. After 48h, the cells were super-transfected with RunX2-luciferase and Renilla-luciferase alone, or in the presence of the PC1-CTT. Luciferase values were measured 24 h after RunX2-lucferase transfection (72 h total after initial shRNA treatment). (C) HEK293 cells were transfected with Myc-p300, FLAG-TAZ, and HA-PC1-CTT where indicated. Cell lysates were incubated with anti-FLAG beads, complexes were eluted in SDS-PAGE loading buffer and run on a 10% SDS- polyacrylamide gel and blotted with the indicated antibodies. Densitometry was performed on the immunoprecipitated p300 band using image analysis software (right panel). Results are expressed as mean ± SE from four independent experiments.

    Techniques Used: Activation Assay, Transfection, shRNA, Quantitative RT-PCR, Luciferase, Incubation, SDS Page, Immunoprecipitation, Software

    Expression of the PC1-CTT does not affect TAZ nuclear localization. HEK293 cells were transfected with FLAG-TAZ(WT) and FLAG-TAZ(S89A) alone or co-transfected with HA-PC1-CTT. The cells were fixed and stained with anti-FLAG (green) and anti-HA (red) antibodies and with DAPI (blue) to reveal the locations of nuclei.
    Figure Legend Snippet: Expression of the PC1-CTT does not affect TAZ nuclear localization. HEK293 cells were transfected with FLAG-TAZ(WT) and FLAG-TAZ(S89A) alone or co-transfected with HA-PC1-CTT. The cells were fixed and stained with anti-FLAG (green) and anti-HA (red) antibodies and with DAPI (blue) to reveal the locations of nuclei.

    Techniques Used: Expressing, Transfection, Staining

    PC1-CTT enhances the TAZ-RunX2 association. (A) HEK293 cells were transfected with RunX2, TAZ and HA-PC1-CTT where indicated. Cell lysates were incubated with anti- TAZ antibody, complexes were eluted in SDS-PAGE loading buffer and run on a 10% SDS- polyacrylamide gel and blotted with the indicated antibodies. Densitometry was performed on the immunoprecipitated RunX2 band using image analysis software (right panel) (OE = Over Expressed). Results are expressed as mean ± SE from four independent experiments. (B) HEK293 cells were transfected with RunX2, TAZ and HA-PC1-CTT where indicated. Cell lysates were incubated with anti-RunX2 antibody, complexes were eluted in SDS-PAGE loading buffer and run on a 10% SDS- polyacrylamide gel and blotted with the indicated antibodies. Densitometry was performed on the immunoprecipitated TAZ band using image analysis software (right panel). Results are expressed as mean ± SE from four independent experiments.
    Figure Legend Snippet: PC1-CTT enhances the TAZ-RunX2 association. (A) HEK293 cells were transfected with RunX2, TAZ and HA-PC1-CTT where indicated. Cell lysates were incubated with anti- TAZ antibody, complexes were eluted in SDS-PAGE loading buffer and run on a 10% SDS- polyacrylamide gel and blotted with the indicated antibodies. Densitometry was performed on the immunoprecipitated RunX2 band using image analysis software (right panel) (OE = Over Expressed). Results are expressed as mean ± SE from four independent experiments. (B) HEK293 cells were transfected with RunX2, TAZ and HA-PC1-CTT where indicated. Cell lysates were incubated with anti-RunX2 antibody, complexes were eluted in SDS-PAGE loading buffer and run on a 10% SDS- polyacrylamide gel and blotted with the indicated antibodies. Densitometry was performed on the immunoprecipitated TAZ band using image analysis software (right panel). Results are expressed as mean ± SE from four independent experiments.

    Techniques Used: Transfection, Incubation, SDS Page, Immunoprecipitation, Software

    TAZ and RunX2 activities are stimulated by the PC1-CTT. (A) HEK293 cells were transfected with TAZ-Gal4, UAS-Luciferase and Renilla luciferase reporter constructs alone or in the presence of the PC1-CTT or PC1-CTT∆NLS and luciferase activity was measured 24 h later. (B) Pkd1 flox/- and Pkd1 -/- cells stably expressing HA-PC1-CTT in a TET-Off inducible vector were transfected with RunX2-luciferase and Renilla reporter constructs in the presence or absence of doxycyclin to induce expression of the PC1-CTT and luciferase activity was measured 24 h later. (C) C3H10T 1/2 cells were reverse transfected (2× serial transfections) with either siControl (non-targeting RNA) or siRNA directed against mouse Pkd1 . mRNA knockdown efficiency was assessed by qRT-PCR after 72 hrs. (D) C3H10T 1/2 cells were first reverse-transfected with either siControl or si Pkd1 RNAi, then super-transfected with RunX2-luciferase and Renilla-luciferase alone or in the presence of the PC1-CTT. Luciferase values were measured 24 h after RunX2-lucferase transfection (72 h total after initial siRNA treatment). Results are expressed as mean ± standard error (SE) from nine biological replicates of three independent experiments.
    Figure Legend Snippet: TAZ and RunX2 activities are stimulated by the PC1-CTT. (A) HEK293 cells were transfected with TAZ-Gal4, UAS-Luciferase and Renilla luciferase reporter constructs alone or in the presence of the PC1-CTT or PC1-CTT∆NLS and luciferase activity was measured 24 h later. (B) Pkd1 flox/- and Pkd1 -/- cells stably expressing HA-PC1-CTT in a TET-Off inducible vector were transfected with RunX2-luciferase and Renilla reporter constructs in the presence or absence of doxycyclin to induce expression of the PC1-CTT and luciferase activity was measured 24 h later. (C) C3H10T 1/2 cells were reverse transfected (2× serial transfections) with either siControl (non-targeting RNA) or siRNA directed against mouse Pkd1 . mRNA knockdown efficiency was assessed by qRT-PCR after 72 hrs. (D) C3H10T 1/2 cells were first reverse-transfected with either siControl or si Pkd1 RNAi, then super-transfected with RunX2-luciferase and Renilla-luciferase alone or in the presence of the PC1-CTT. Luciferase values were measured 24 h after RunX2-lucferase transfection (72 h total after initial siRNA treatment). Results are expressed as mean ± standard error (SE) from nine biological replicates of three independent experiments.

    Techniques Used: Transfection, Luciferase, Construct, Activity Assay, Stable Transfection, Expressing, Plasmid Preparation, Quantitative RT-PCR

    36) Product Images from "CHCHD2 accumulates in distressed mitochondria and facilitates oligomerization of CHCHD10"

    Article Title: CHCHD2 accumulates in distressed mitochondria and facilitates oligomerization of CHCHD10

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddy270

    CHCHD2 and CHCHD10 localize to mitochondria cristae and are not essential for MICOS complex stability. (A) Representative Immuno-TEM images labelled pre-embedding with MIC19/CHCHD3, CHCHD2 or CHCHD10 antibodies followed by nanogold-conjugated secondary antibodies. (B) Histograms depicting distance from individual intra-mitochondrial nanogold particles to the nearest cristae junction (CJ) for MIC19/CHCHD3 (top), CHCHD2 (middle) and CHCHD10 (bottom). Particles in the cytosol or associated with the cytosolic face of the outer membrane were excluded from the analysis. (C) Representative TEM images from Doxycycline (Dox) inducible knockdown HeLa cells targeting MIC60 (MIC60 KD), WT HEK293 cells and C2/10 DKO cell lines. Dox-inducible cell lines were treated with 2 μm Dox for 7 days (Dox+) or left untreated (Dox−). Greater than 200 mitochondria were scored per condition. (D) Model of MICOS complex relative to the intermembrane space (IMS) and inner membrane composed of the inner boundary membrane (IBM), CJ and cristae. (E) Lysates from MIC60 KD HeLa cells +/− Dox were immunoblotted with MIC60, MIC25/CHCHD6, CHCHD2, MIC19/CHCHD3, CHCHD10 and Tubulin antibodies. Tubulin served as a loading control. (F) Lysates from WT, C2 KO, C10 KO, C2/10 DKO HEK293 cells were immunoblotted with the same antibodies as in (E).
    Figure Legend Snippet: CHCHD2 and CHCHD10 localize to mitochondria cristae and are not essential for MICOS complex stability. (A) Representative Immuno-TEM images labelled pre-embedding with MIC19/CHCHD3, CHCHD2 or CHCHD10 antibodies followed by nanogold-conjugated secondary antibodies. (B) Histograms depicting distance from individual intra-mitochondrial nanogold particles to the nearest cristae junction (CJ) for MIC19/CHCHD3 (top), CHCHD2 (middle) and CHCHD10 (bottom). Particles in the cytosol or associated with the cytosolic face of the outer membrane were excluded from the analysis. (C) Representative TEM images from Doxycycline (Dox) inducible knockdown HeLa cells targeting MIC60 (MIC60 KD), WT HEK293 cells and C2/10 DKO cell lines. Dox-inducible cell lines were treated with 2 μm Dox for 7 days (Dox+) or left untreated (Dox−). Greater than 200 mitochondria were scored per condition. (D) Model of MICOS complex relative to the intermembrane space (IMS) and inner membrane composed of the inner boundary membrane (IBM), CJ and cristae. (E) Lysates from MIC60 KD HeLa cells +/− Dox were immunoblotted with MIC60, MIC25/CHCHD6, CHCHD2, MIC19/CHCHD3, CHCHD10 and Tubulin antibodies. Tubulin served as a loading control. (F) Lysates from WT, C2 KO, C10 KO, C2/10 DKO HEK293 cells were immunoblotted with the same antibodies as in (E).

    Techniques Used: Transmission Electron Microscopy

    CHCHD2 and CHCHD10 are stabilized in mitochondria post-translationally following loss of Δψ. (A) Representative confocal images of WT HeLa cells treated for 24 h with dimethyl sulfoxide (DMSO) or CCCP (10 μM) and immunostained with CHCHD2 (green) and cytochrome c (red) antibodies and incubated with 4',6-diamidino-2-phenylindole (DAPI) to label nuclei (blue). Cytochrome c was used as a mitochondrial marker. Retention of cytochrome c in mitochondria also indicates that the cells analyzed have not undergone apoptosis after 24 h CCCP treatment, as cytochrome c is released from mitochondria during apoptosis. (B) CHCHD2 intensity in regions overlapping with DAPI and cytochrome c, representing nuclear and mitochondrial compartments, respectively, was measured in 11 fields from three biological replicates for each condition in cells treated as described in (A). (C) Lysates from WT HeLa cells stably expressing CHCHD2 WT-Flag or CHCHD2 T61I-Flag and treated with DMSO or CCCP (10 μm) for 24 h were immunoblotted (IB) using CHCHD2 and Tubulin antibodies. Tubulin was used as a loading control. (D) Table summarizes changes in CHCHD2 protein and mRNA expression in response to mitochondrial stressors from previously published datasets. CI KO with resp dys, average fold change resulting from KO of complex I supernumerary subunits that cause respiratory dysfunction; all other, fold change of CHCHD2 protein level following FCCP treatment versus average of control, actinonin treatment, doxycycline treatment and MitoBloCK-6 treatment. All treatments were for 24 h (E) Lysates from WT and PINK1 KO HeLa cells treated with CCCP (C) or DMSO (D) for 6 h were immunoblotted for GAPDH, CHCHD2 and PINK1. The * indicates non-specific band. (F) Lysates from WT and C2/10 DKO HEK293 cells were immunoblotted with GAPDH, CHCHD2, PINK1 and Mfn1. (G) Lysates from WT HeLa cells treated overnight with DMSO (D) or CCCP (C) followed by the addition of CHX for 0–6 h were immunoblotted with CHCHD2, CHCHD10 and GAPDH antibodies. The * indicates misloaded well. (H) Quantification of CHCHD2 and CHCHD10 protein levels from experiment shown in (G) from > = 3 biological replicates performed on at least two separate occasions CHCHD2 and CHCHD10 protein levels at each time point were normalized to their value at time point 0 hr.
    Figure Legend Snippet: CHCHD2 and CHCHD10 are stabilized in mitochondria post-translationally following loss of Δψ. (A) Representative confocal images of WT HeLa cells treated for 24 h with dimethyl sulfoxide (DMSO) or CCCP (10 μM) and immunostained with CHCHD2 (green) and cytochrome c (red) antibodies and incubated with 4',6-diamidino-2-phenylindole (DAPI) to label nuclei (blue). Cytochrome c was used as a mitochondrial marker. Retention of cytochrome c in mitochondria also indicates that the cells analyzed have not undergone apoptosis after 24 h CCCP treatment, as cytochrome c is released from mitochondria during apoptosis. (B) CHCHD2 intensity in regions overlapping with DAPI and cytochrome c, representing nuclear and mitochondrial compartments, respectively, was measured in 11 fields from three biological replicates for each condition in cells treated as described in (A). (C) Lysates from WT HeLa cells stably expressing CHCHD2 WT-Flag or CHCHD2 T61I-Flag and treated with DMSO or CCCP (10 μm) for 24 h were immunoblotted (IB) using CHCHD2 and Tubulin antibodies. Tubulin was used as a loading control. (D) Table summarizes changes in CHCHD2 protein and mRNA expression in response to mitochondrial stressors from previously published datasets. CI KO with resp dys, average fold change resulting from KO of complex I supernumerary subunits that cause respiratory dysfunction; all other, fold change of CHCHD2 protein level following FCCP treatment versus average of control, actinonin treatment, doxycycline treatment and MitoBloCK-6 treatment. All treatments were for 24 h (E) Lysates from WT and PINK1 KO HeLa cells treated with CCCP (C) or DMSO (D) for 6 h were immunoblotted for GAPDH, CHCHD2 and PINK1. The * indicates non-specific band. (F) Lysates from WT and C2/10 DKO HEK293 cells were immunoblotted with GAPDH, CHCHD2, PINK1 and Mfn1. (G) Lysates from WT HeLa cells treated overnight with DMSO (D) or CCCP (C) followed by the addition of CHX for 0–6 h were immunoblotted with CHCHD2, CHCHD10 and GAPDH antibodies. The * indicates misloaded well. (H) Quantification of CHCHD2 and CHCHD10 protein levels from experiment shown in (G) from > = 3 biological replicates performed on at least two separate occasions CHCHD2 and CHCHD10 protein levels at each time point were normalized to their value at time point 0 hr.

    Techniques Used: Incubation, Marker, Stable Transfection, Expressing

    Disease-causing mutations cluster in conserved GXXXGXXXG motif and cause protein aggregation. (A) Position of disease-causing mutations (green) is indicated relative to conserved GXXXGXXXG motif (red). (B) Helical wheel representation of CHCHD2 sequence modeled with 3.9 amino acids per turn, as is classically observed for GXXXG motif containing helices. Position of disease mutations in CHCHD2 and CHCHD10 is indicated by green arrowheads. (C and D) Triton-X100 soluble and insoluble fractions from WT HEK293 cells transiently transfected with CHCHD2 WT, CHCHD2 T61I, CHCHD10 G58R, CHCHD10 S59L and CHCHD10 G66V or CHCHD10 G58R, G66V cDNA were immunoblotted for CHCHD2, CHCHD10 and Tubulin. Tubulin from the soluble fraction was used as a loading control. (E) Representative images from WT ( top panels) or CHCHD2 KO (bottom panels) HeLa cells were transiently transfected with C-terminal Flag tagged CHCHD10 WT, CHCHD10 G58R, CHCHD10 S59L, CHCHD10 G66V and immunostained with Flag and Tom20 antibodies. Tom20 was used as a mitochondrial marker. (F) Percentage of cells with indicated mitochondrial phenotypes in cells treated as described in (E) from > = 3 biological replicates performed on at least two separate occasions.
    Figure Legend Snippet: Disease-causing mutations cluster in conserved GXXXGXXXG motif and cause protein aggregation. (A) Position of disease-causing mutations (green) is indicated relative to conserved GXXXGXXXG motif (red). (B) Helical wheel representation of CHCHD2 sequence modeled with 3.9 amino acids per turn, as is classically observed for GXXXG motif containing helices. Position of disease mutations in CHCHD2 and CHCHD10 is indicated by green arrowheads. (C and D) Triton-X100 soluble and insoluble fractions from WT HEK293 cells transiently transfected with CHCHD2 WT, CHCHD2 T61I, CHCHD10 G58R, CHCHD10 S59L and CHCHD10 G66V or CHCHD10 G58R, G66V cDNA were immunoblotted for CHCHD2, CHCHD10 and Tubulin. Tubulin from the soluble fraction was used as a loading control. (E) Representative images from WT ( top panels) or CHCHD2 KO (bottom panels) HeLa cells were transiently transfected with C-terminal Flag tagged CHCHD10 WT, CHCHD10 G58R, CHCHD10 S59L, CHCHD10 G66V and immunostained with Flag and Tom20 antibodies. Tom20 was used as a mitochondrial marker. (F) Percentage of cells with indicated mitochondrial phenotypes in cells treated as described in (E) from > = 3 biological replicates performed on at least two separate occasions.

    Techniques Used: Sequencing, Transfection, Marker

    CHCHD2 and CHCHD10 are functionally redundant. (A) Lysates from wildtype (WT), CHCHD2 knockout (C2 KO), CHCHD10 knockout (C10 KO) or CHCHD2/10 DKO (C2/10 DKO) HEK293 cells were immunoblotted with CHCHD2, CHCHD10, COX2 and Tubulin antibodies. Tubulin served as a loading control. (B) Quantification of (A) from > = 3 biological replicates performed on at least 2 separate occasions. (C) Quantification of relative cytosolic oxidation of HEK293 WT, C2 KO, C10 KO and DKO cells using roGFP probe. (D) Oxygen consumption rate (OCR) of WT and C2/10 DKO HEK293 cell lines incubated in glucose (left panel) or galactose (right panel). (E) Spectrophotometric analysis of mitochondrial respiratory chain activities. COX, (complex IV); SCCR, succinate cytochrome c reductase (complex II and III); GPCCR, glycerol-3-phosphate cytochrome c reductase; QCCR, ubiquinol cytochrome c reductase (complex III). Samples were tested in duplicate and average values reported. (F) Representative confocal images of WT, C2 KO and C10 KO HeLa cells immunostained with antibodies against endogenous C2, C10 and MIC19/CHCHD3. MIC19/CHCHD3 staining was used as a mitochondrial marker. (G) Representative confocal and STED images of HeLa cells immunostained with a CHCHD10 antibody (top panels). Representative STED image of HeLa cells immunostained with CHCHD2 and CHCHD10 antibodies (bottom panels).
    Figure Legend Snippet: CHCHD2 and CHCHD10 are functionally redundant. (A) Lysates from wildtype (WT), CHCHD2 knockout (C2 KO), CHCHD10 knockout (C10 KO) or CHCHD2/10 DKO (C2/10 DKO) HEK293 cells were immunoblotted with CHCHD2, CHCHD10, COX2 and Tubulin antibodies. Tubulin served as a loading control. (B) Quantification of (A) from > = 3 biological replicates performed on at least 2 separate occasions. (C) Quantification of relative cytosolic oxidation of HEK293 WT, C2 KO, C10 KO and DKO cells using roGFP probe. (D) Oxygen consumption rate (OCR) of WT and C2/10 DKO HEK293 cell lines incubated in glucose (left panel) or galactose (right panel). (E) Spectrophotometric analysis of mitochondrial respiratory chain activities. COX, (complex IV); SCCR, succinate cytochrome c reductase (complex II and III); GPCCR, glycerol-3-phosphate cytochrome c reductase; QCCR, ubiquinol cytochrome c reductase (complex III). Samples were tested in duplicate and average values reported. (F) Representative confocal images of WT, C2 KO and C10 KO HeLa cells immunostained with antibodies against endogenous C2, C10 and MIC19/CHCHD3. MIC19/CHCHD3 staining was used as a mitochondrial marker. (G) Representative confocal and STED images of HeLa cells immunostained with a CHCHD10 antibody (top panels). Representative STED image of HeLa cells immunostained with CHCHD2 and CHCHD10 antibodies (bottom panels).

    Techniques Used: Knock-Out, Incubation, Staining, Marker

    37) Product Images from "Asparagine-linked glycosylation is not directly coupled to protein translocation across the endoplasmic reticulum in Saccharomyces cerevisiae"

    Article Title: Asparagine-linked glycosylation is not directly coupled to protein translocation across the endoplasmic reticulum in Saccharomyces cerevisiae

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E19-06-0330

    Pulse labeling of SHBG in human and yeast cells. (A) Diagram of SHBG showing signal sequences for expression of SHBG in human and yeast cells, the location of disulfide bonds, and the C-terminal glycosylation sites. (B) Pulse–chase labeling of SHBG in wild-type and mutant HEK293 cells (left panel, 5 min pulse, 20 min chase). Pulse labeling of CPYss-SHBG for 8 min in yeast cells (right panel). (C) Pulse–chase labeling of CPYss-SHBG in yeast using the indicated pulse (P) and chase (C) intervals. In B and C, quantified values are of the experiment shown, which is representative of two replicates. EH indicates digestion with endoglycosidase H.
    Figure Legend Snippet: Pulse labeling of SHBG in human and yeast cells. (A) Diagram of SHBG showing signal sequences for expression of SHBG in human and yeast cells, the location of disulfide bonds, and the C-terminal glycosylation sites. (B) Pulse–chase labeling of SHBG in wild-type and mutant HEK293 cells (left panel, 5 min pulse, 20 min chase). Pulse labeling of CPYss-SHBG for 8 min in yeast cells (right panel). (C) Pulse–chase labeling of CPYss-SHBG in yeast using the indicated pulse (P) and chase (C) intervals. In B and C, quantified values are of the experiment shown, which is representative of two replicates. EH indicates digestion with endoglycosidase H.

    Techniques Used: Labeling, Expressing, Pulse Chase, Mutagenesis

    Glycosylation of human prosaposin in yeast and human cells. (A) SILAC-based glycoproteomic analysis of prosaposin glycosylation in HEK293-derived cells that lack either the STT3A or STT3B complex. Site occupancy is expressed as Δlog 2 , where a negative value indicates reduced glycosylation in the mutant cells relative to the wild-type cells. Error bars designate standard deviations ( n = 3–7) or individual data points (*, n = 2). (B) Signal sequences for wild-type CPY and a more hydrophobic derivative (CPY+4). The underlined leucine residues in CPY+4 replace marginally hydrophobic amino acids. The diagonal line designates the signal sequence cleavage site. (C) CPY and pSAP constructs that have the CPY signal sequence or the CPY+4 signal sequence were pulse-labeled for 7 min with Tran- 35 S label in wild-type yeast cells. (D) Diagrams of the SAP, SAPΔ3,5, and SAPΔ1,2,4 constructs for expression of prosaposin derivatives in yeast and human cells. The signal sequences for human prosaposin (SAP ss ) and yeast CPY (CPY ss ) are for expression in HEK293 cells and yeast, respectively. Saposin domains are designated by cyan rectangles. Red lines designate disulfides. The disulfide bonding pattern of the N-terminal and C-terminal flanking domains is not known. A C-terminal DDK-His tag was appended for immunoprecipitation with anti-DDK sera. (E) Pulse labeling (10 min) of pSAP, pSAPΔ1,2,4, and pSAPΔ3,5 in wild-type and STT3A-deficient HEK293 cells. (F) Pulse labeling (7 min) of SAP, SAPΔ3,5, and SAPΔ1,2,4 in yeast. (G) Pulse–chase labeling of CPYss-SAP (Δ1,2,4) in yeast using the indicated pulse (P) and chase (C) intervals. (F, G) Quantified values are of the experiment shown, which is representative of two or more replicates. In C and E–G, labeled arrows indicate the number of glycans. EH indicates digestion with endoglycosidase H.
    Figure Legend Snippet: Glycosylation of human prosaposin in yeast and human cells. (A) SILAC-based glycoproteomic analysis of prosaposin glycosylation in HEK293-derived cells that lack either the STT3A or STT3B complex. Site occupancy is expressed as Δlog 2 , where a negative value indicates reduced glycosylation in the mutant cells relative to the wild-type cells. Error bars designate standard deviations ( n = 3–7) or individual data points (*, n = 2). (B) Signal sequences for wild-type CPY and a more hydrophobic derivative (CPY+4). The underlined leucine residues in CPY+4 replace marginally hydrophobic amino acids. The diagonal line designates the signal sequence cleavage site. (C) CPY and pSAP constructs that have the CPY signal sequence or the CPY+4 signal sequence were pulse-labeled for 7 min with Tran- 35 S label in wild-type yeast cells. (D) Diagrams of the SAP, SAPΔ3,5, and SAPΔ1,2,4 constructs for expression of prosaposin derivatives in yeast and human cells. The signal sequences for human prosaposin (SAP ss ) and yeast CPY (CPY ss ) are for expression in HEK293 cells and yeast, respectively. Saposin domains are designated by cyan rectangles. Red lines designate disulfides. The disulfide bonding pattern of the N-terminal and C-terminal flanking domains is not known. A C-terminal DDK-His tag was appended for immunoprecipitation with anti-DDK sera. (E) Pulse labeling (10 min) of pSAP, pSAPΔ1,2,4, and pSAPΔ3,5 in wild-type and STT3A-deficient HEK293 cells. (F) Pulse labeling (7 min) of SAP, SAPΔ3,5, and SAPΔ1,2,4 in yeast. (G) Pulse–chase labeling of CPYss-SAP (Δ1,2,4) in yeast using the indicated pulse (P) and chase (C) intervals. (F, G) Quantified values are of the experiment shown, which is representative of two or more replicates. In C and E–G, labeled arrows indicate the number of glycans. EH indicates digestion with endoglycosidase H.

    Techniques Used: Derivative Assay, Mutagenesis, Sequencing, Construct, Labeling, Expressing, Immunoprecipitation, Pulse Chase

    Glycosylation of CPY derivatives in yeast and human cells. (A) Diagram of CPY derivatives with closely spaced acceptor sites. The wild-type signal sequence of CPY was replaced with either the prosaposin signal sequence (SAP ss ) for expression in human cells or the CPY+4 sequence to direct cotranslational translocation in yeast. The four glycosylation sites in CPY were eliminated by the indicated N to Q mutations to create the SAPss-CPYΔ4 and CPY+4ss-CPYΔ4 constructs. Two or three closely spaced NXS or NXT sites were created at the indicated sites to yield eight reporters. The introduced glycosylation sites are underlined. (B) Pulse labeling of the SAP ss -CPY and SAP ss -CPYΔ4 derivatives in wild-type and STT3A(–/–) HEK293 cells. Downward-pointing arrowheads indicate the fully glycosylated form of the T12 and T123 reporters. Upward-pointing arrowheads indicate hypoglycosylated forms of S12 and S45 reporter that were detected in STT3A(–/–) cells. (C) Pulse labeling (4 min) of CPY+4 ss -CPY and CPY+4 ss -CPYΔ4 derivatives expressed in yeast. Upward-pointing arrowheads designate hypoglycosylated forms of the reporters that are either less abundant or not detected in wild-type HEK293 cells. Note the absence of the fully glycosylated T12 and T123 reporters. (D) Pulse–chase labeling of selected CPY+4 ss -CPYΔ4 derivatives in yeast. The pulse period was 2 min for all samples. Upward-pointing arrowheads designate hypoglycosylated forms of NXS containing reporters that are more prominent before the chase period. The downward-pointing arrowheads designate trace amounts of the fully glycosylated T12 and T123 reporters that were visible after the chase. The quantified values below the gel lanes are for the experiment shown, which is representative of two similar experiments.
    Figure Legend Snippet: Glycosylation of CPY derivatives in yeast and human cells. (A) Diagram of CPY derivatives with closely spaced acceptor sites. The wild-type signal sequence of CPY was replaced with either the prosaposin signal sequence (SAP ss ) for expression in human cells or the CPY+4 sequence to direct cotranslational translocation in yeast. The four glycosylation sites in CPY were eliminated by the indicated N to Q mutations to create the SAPss-CPYΔ4 and CPY+4ss-CPYΔ4 constructs. Two or three closely spaced NXS or NXT sites were created at the indicated sites to yield eight reporters. The introduced glycosylation sites are underlined. (B) Pulse labeling of the SAP ss -CPY and SAP ss -CPYΔ4 derivatives in wild-type and STT3A(–/–) HEK293 cells. Downward-pointing arrowheads indicate the fully glycosylated form of the T12 and T123 reporters. Upward-pointing arrowheads indicate hypoglycosylated forms of S12 and S45 reporter that were detected in STT3A(–/–) cells. (C) Pulse labeling (4 min) of CPY+4 ss -CPY and CPY+4 ss -CPYΔ4 derivatives expressed in yeast. Upward-pointing arrowheads designate hypoglycosylated forms of the reporters that are either less abundant or not detected in wild-type HEK293 cells. Note the absence of the fully glycosylated T12 and T123 reporters. (D) Pulse–chase labeling of selected CPY+4 ss -CPYΔ4 derivatives in yeast. The pulse period was 2 min for all samples. Upward-pointing arrowheads designate hypoglycosylated forms of NXS containing reporters that are more prominent before the chase period. The downward-pointing arrowheads designate trace amounts of the fully glycosylated T12 and T123 reporters that were visible after the chase. The quantified values below the gel lanes are for the experiment shown, which is representative of two similar experiments.

    Techniques Used: Sequencing, Expressing, Translocation Assay, Construct, Labeling, Pulse Chase

    38) Product Images from "The Administration of Cortisol Induces Female-to-Male Sex Change in the Protogynous Orange-Spotted Grouper, Epinephelus coioides"

    Article Title: The Administration of Cortisol Induces Female-to-Male Sex Change in the Protogynous Orange-Spotted Grouper, Epinephelus coioides

    Journal: Frontiers in Endocrinology

    doi: 10.3389/fendo.2020.00012

    Luciferase activity in response to cortisol-stimulation in vitro receptor binding assay. (A) Overexpression of orange-spotted grouper GRs (GR1 and GR2) in HEK293 cells. Western blotting experiments were performed, and anti-his tag antibody was used. M indicates molecular marker in kDa. (B) Different cortisol concentrations induced GRE-driven luciferase activity in HEK293 cells. The construct overexpressing orange-spotted grouper GR1 or GR2 was transfected into HEK293 cells with a luciferase reporter gene (GRE-Luc). An empty construct without GR overexpression was used as the control. The cells were harvested for luciferase activity analysis after 12 h hormone treatment. (C) Effect of cortisol on the activity of the cyp19a1a promoter. The construct overexpressing GR1 or GR2 was transfected into HEK293 cells with cyp19a1a promoter construct. An empty construct without GR overexpression was transfected into HEK293 cells, with cyp19a1a promoter construct as the control. (D) Effect of cortisol on the activity of the amh promoter. The construct overexpressing GR1 or GR2 was transfected into HEK293 cells with amh promoter construct. An empty construct without GR overexpression was transfected into HEK293 cells, with amh promoter construct as the control. The cells were harvested for luciferase activity analysis after 12 h cortisol (1,000 ng/ml) treatment. Data are presented as the mean of four replicate wells ± SEM. The experiments were independently repeated three times. The asterisk ** indicates statistically significant difference ( P
    Figure Legend Snippet: Luciferase activity in response to cortisol-stimulation in vitro receptor binding assay. (A) Overexpression of orange-spotted grouper GRs (GR1 and GR2) in HEK293 cells. Western blotting experiments were performed, and anti-his tag antibody was used. M indicates molecular marker in kDa. (B) Different cortisol concentrations induced GRE-driven luciferase activity in HEK293 cells. The construct overexpressing orange-spotted grouper GR1 or GR2 was transfected into HEK293 cells with a luciferase reporter gene (GRE-Luc). An empty construct without GR overexpression was used as the control. The cells were harvested for luciferase activity analysis after 12 h hormone treatment. (C) Effect of cortisol on the activity of the cyp19a1a promoter. The construct overexpressing GR1 or GR2 was transfected into HEK293 cells with cyp19a1a promoter construct. An empty construct without GR overexpression was transfected into HEK293 cells, with cyp19a1a promoter construct as the control. (D) Effect of cortisol on the activity of the amh promoter. The construct overexpressing GR1 or GR2 was transfected into HEK293 cells with amh promoter construct. An empty construct without GR overexpression was transfected into HEK293 cells, with amh promoter construct as the control. The cells were harvested for luciferase activity analysis after 12 h cortisol (1,000 ng/ml) treatment. Data are presented as the mean of four replicate wells ± SEM. The experiments were independently repeated three times. The asterisk ** indicates statistically significant difference ( P

    Techniques Used: Luciferase, Activity Assay, In Vitro, Reporter Assay, Over Expression, Western Blot, Marker, Construct, Transfection

    39) Product Images from "HSP90A inhibition promotes anti-tumor immunity by reversing multi-modal resistance and stem-like property of immune-refractory tumors"

    Article Title: HSP90A inhibition promotes anti-tumor immunity by reversing multi-modal resistance and stem-like property of immune-refractory tumors

    Journal: Nature Communications

    doi: 10.1038/s41467-019-14259-y

    HSP90AA1 expression is directly regulated by NANOG. a and b CaSki P3 cells were transfected with siRNA-targeting GFP or NANOG. a Levels of NANOG and HSP90A protein were analyzed by Western blot. b HSP90AA1 mRNA expression was analyzed by qRT-PCR. c and d CaSki P0 cells were stably transfected with empty vector (no insert) or NANOG. c Levels of NANOG and HSP90A protein were analyzed by Western blot. d HSP90AA1 mRNA expression was analyzed by qRT-PCR. e and f HEK293 cells were transfected with empty vector (no insert), FLAG-NANOG wild type (NANOG WT) or FLAG-NANOG mutant (NANOG MUT). e Levels of HSP90A and FLAG-NANOG proteins were proved by Western blot. f HSP90AA1 mRNA expression was analyzed by qRT-PCR. g Diagram of HSP90AA1 promoter region (−1322 to +190) containing NANOG binding element. The arrows indicate ChIP amplicon corresponding to −1048 to −899. h Luciferase assay in HEK293 cells transfected with the pGL3-HSP90AA1 WT or MUT plasmid, together no insert, NANOG WT or NANOG MUT plasmids. i Chromatin immunoprecipitation assay was carried out using HEK293 cells transfected with FLAG-NANOG. Cross-linked chromatin was immunoprecipitated with anti-FLAG antibodies. Immunoprecipitated DNAs were amplified with PCR primers specific for the HSP90AA1 promoter region indicated above. Mouse IgG was used as a negative control. The input represents 2% of the total chromatin. j ChIP assay was carried out using CaSki P0 and P3 cells. Cross-linked chromatin was immunoprecipitated with anti-NANOG antibodies. The value of ChIP data represent relative ratio to the input. β-ACTIN was included as an internal loading control. Numbers below blot images indicate the expression as measured by fold change a , c , and e . All experiments were performed in triplicate. The p -values by two-tailed Student’s t test b , d and j , one-way ANOVA f or two-way ANOVA h are indicated. Data represent the mean ± SD. Source data are provided as a Source Data file.
    Figure Legend Snippet: HSP90AA1 expression is directly regulated by NANOG. a and b CaSki P3 cells were transfected with siRNA-targeting GFP or NANOG. a Levels of NANOG and HSP90A protein were analyzed by Western blot. b HSP90AA1 mRNA expression was analyzed by qRT-PCR. c and d CaSki P0 cells were stably transfected with empty vector (no insert) or NANOG. c Levels of NANOG and HSP90A protein were analyzed by Western blot. d HSP90AA1 mRNA expression was analyzed by qRT-PCR. e and f HEK293 cells were transfected with empty vector (no insert), FLAG-NANOG wild type (NANOG WT) or FLAG-NANOG mutant (NANOG MUT). e Levels of HSP90A and FLAG-NANOG proteins were proved by Western blot. f HSP90AA1 mRNA expression was analyzed by qRT-PCR. g Diagram of HSP90AA1 promoter region (−1322 to +190) containing NANOG binding element. The arrows indicate ChIP amplicon corresponding to −1048 to −899. h Luciferase assay in HEK293 cells transfected with the pGL3-HSP90AA1 WT or MUT plasmid, together no insert, NANOG WT or NANOG MUT plasmids. i Chromatin immunoprecipitation assay was carried out using HEK293 cells transfected with FLAG-NANOG. Cross-linked chromatin was immunoprecipitated with anti-FLAG antibodies. Immunoprecipitated DNAs were amplified with PCR primers specific for the HSP90AA1 promoter region indicated above. Mouse IgG was used as a negative control. The input represents 2% of the total chromatin. j ChIP assay was carried out using CaSki P0 and P3 cells. Cross-linked chromatin was immunoprecipitated with anti-NANOG antibodies. The value of ChIP data represent relative ratio to the input. β-ACTIN was included as an internal loading control. Numbers below blot images indicate the expression as measured by fold change a , c , and e . All experiments were performed in triplicate. The p -values by two-tailed Student’s t test b , d and j , one-way ANOVA f or two-way ANOVA h are indicated. Data represent the mean ± SD. Source data are provided as a Source Data file.

    Techniques Used: Expressing, Transfection, Western Blot, Quantitative RT-PCR, Stable Transfection, Plasmid Preparation, Mutagenesis, Binding Assay, Chromatin Immunoprecipitation, Amplification, Luciferase, Immunoprecipitation, Polymerase Chain Reaction, Negative Control, Two Tailed Test

    40) Product Images from "Histone H4 Lys 20 monomethylation by histone methylase SET8 mediates Wnt target gene activation"

    Article Title: Histone H4 Lys 20 monomethylation by histone methylase SET8 mediates Wnt target gene activation

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

    doi: 10.1073/pnas.1009353108

    Wnt/β-catenin facilitates SET8/TCF4 complex formation. ( A ) Overexpression β-catenin facilitated endogenous SET8 augmentation on the AXIN2 promoter. HEK293 cells were transfected with ΔN-β-catenin or LacZ, and ChIP experiments
    Figure Legend Snippet: Wnt/β-catenin facilitates SET8/TCF4 complex formation. ( A ) Overexpression β-catenin facilitated endogenous SET8 augmentation on the AXIN2 promoter. HEK293 cells were transfected with ΔN-β-catenin or LacZ, and ChIP experiments

    Techniques Used: Over Expression, Transfection, Chromatin Immunoprecipitation

    SET8 interacts with the TCF4 family. ( A and B ) Coimmunoprecipitation of SET8 and TCF4/LEF1 in HEK293T cells. ( C ) Endogenous SET8 and TCF4 could form a complex under Wnt3a stimulation. HEK293 cells were treated with control or Wnt3a CM for 1 h. ( D ) Schematic
    Figure Legend Snippet: SET8 interacts with the TCF4 family. ( A and B ) Coimmunoprecipitation of SET8 and TCF4/LEF1 in HEK293T cells. ( C ) Endogenous SET8 and TCF4 could form a complex under Wnt3a stimulation. HEK293 cells were treated with control or Wnt3a CM for 1 h. ( D ) Schematic

    Techniques Used:

    H4K20me-1 is a transcriptional activation marker at the promoter of the Wnt target gene. ( A ) ChIP screen for histone methylation variation on AXIN2 TBE. HEK293 cells were treated with control or Wnt3a CM for 1 h before ChIP with different antibodies.
    Figure Legend Snippet: H4K20me-1 is a transcriptional activation marker at the promoter of the Wnt target gene. ( A ) ChIP screen for histone methylation variation on AXIN2 TBE. HEK293 cells were treated with control or Wnt3a CM for 1 h before ChIP with different antibodies.

    Techniques Used: Activation Assay, Marker, Chromatin Immunoprecipitation, Methylation

    Related Articles

    Transfection:

    Article Title: A Genome-Wide Scan for MicroRNA-Related Genetic Variants Associated With Primary Open-Angle Glaucoma
    Article Snippet: .. HEK293 cells (n = 10,000) were plated into 96-well plates and cotransfected with 1 μg pGL3 containing the 3′UTR with either the major or minor allele, miRNA mimic (mirVana Mimics; Thermo Fischer Scientific, Waltham, MA, USA), and a plasmid expressing the Renilla luciferase that served as transfection control, with Lipofectamine RNAiMAX (Invitrogen). .. Luciferase activity was measured with the Dual-Glo Luciferase Assay System according to manufacturer's protocol (Promega).

    Article Title: Toxoplasma gondii cathepsin proteases are undeveloped prominent vaccine antigens against toxoplasmosis
    Article Snippet: .. TgCPB and TgCPL expression in mammalian cells When the cell density reached 80–90%, HEK293 cells were transfected with pBudCE4.1-TgCPB or pBudCE4.1-TgCPL using Lipofectamine™ 2000 reagent (Invitrogen, USA). .. After 24-h incubation, the cells were fixed with cold methanol for 20 min and protein expression was evaluated using an indirect fluorescence antibody test as previously described [ ].

    Article Title: LRRK2 functions as a Wnt signaling scaffold, bridging cytosolic proteins and membrane-localized LRP6
    Article Snippet: .. HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) plus 100 U/ml penicillin G and 100 μg/ml streptomycin and were transfected using Lipofectamine LTX (Invitrogen) at a 2:1 μl transfection reagent to μg DNA ratio. .. SH-SY5Y cells were cultured in a 1:1 mixture of DMEM and F12 Ham's media supplemented with 10% FBS plus penicillin and streptomycin and were transfected using FuGENE 6 (Roche) at a 2.5:1 μl transfection reagent to μg DNA ratio.

    Article Title: Comparative Genomic and Sequence Analysis Provides Insight into the Molecular Functionality of NOD1 and NOD2
    Article Snippet: .. Subcellular fractionation Membrane and cytosolic fractionation of transfected HEK293 cells was performed using a Subcellular Fractionation Kit (Pierce) as per the manufacturer’s instructions. .. An antibody against GAPDH (Abcam) was used to characterize cytosolic fractions.

    Article Title: Lamin B is a target for selective nuclear PQC by BAG3: implication for nuclear envelopathies
    Article Snippet: .. Expression of mutant protein was verified in HEK293 cells by Lipofectamine (Thermo Fisher) transfection. .. The plasmids mCherry-lamin B1-10 and mCherry-lamin A-C-18 were a kind gift from Michael Davidson lab (Addgene plasmid #55069), as were HA-p62 from Qing Zhong lab (Addgene plasmid #28027), and GFP-Ub from Nico Dantuma (Addgene #11928).

    Luciferase:

    Article Title: A Genome-Wide Scan for MicroRNA-Related Genetic Variants Associated With Primary Open-Angle Glaucoma
    Article Snippet: .. HEK293 cells (n = 10,000) were plated into 96-well plates and cotransfected with 1 μg pGL3 containing the 3′UTR with either the major or minor allele, miRNA mimic (mirVana Mimics; Thermo Fischer Scientific, Waltham, MA, USA), and a plasmid expressing the Renilla luciferase that served as transfection control, with Lipofectamine RNAiMAX (Invitrogen). .. Luciferase activity was measured with the Dual-Glo Luciferase Assay System according to manufacturer's protocol (Promega).

    Mutagenesis:

    Article Title: Lamin B is a target for selective nuclear PQC by BAG3: implication for nuclear envelopathies
    Article Snippet: .. Expression of mutant protein was verified in HEK293 cells by Lipofectamine (Thermo Fisher) transfection. .. The plasmids mCherry-lamin B1-10 and mCherry-lamin A-C-18 were a kind gift from Michael Davidson lab (Addgene plasmid #55069), as were HA-p62 from Qing Zhong lab (Addgene plasmid #28027), and GFP-Ub from Nico Dantuma (Addgene #11928).

    Cell Culture:

    Article Title: Expression of N-terminal truncated desmoglein 3 (?NDg3) in epidermis and its role in keratinocyte differentiation
    Article Snippet: .. Immortalized human keratinocytes HaCaT and HEK293 cells were cultured in DMEM supplemented with 10% FBS (Gibco BRL). .. For differentiation of HaCaT, cells were cultured with 1 µM and 0.3 mM calcium for the indicated time points.

    Article Title: Membrane Transporters for Sulfated Steroids in the Human Testis - Cellular Localization, Expression Pattern and Functional Analysis
    Article Snippet: .. Respective HEK293 cells were cultured in DMEM/F-12 medium (LifeTechnologies), supplemented with 10% FCS (Sigma-Aldrich), L-glutamine (4 mM), penicillin (100 units/ml), and streptomycin (100 µg/ml) at 37°C, 5% CO2 , and 95% humidity. .. Immunofluorescence Microscopy of Stably Transfected Cell Lines For immunofluorescence microscopy, cells were seeded on poly-D-lysine coated glass coverslips in 24-well plates with a density of 1×105 cells per well.

    Fractionation:

    Article Title: Comparative Genomic and Sequence Analysis Provides Insight into the Molecular Functionality of NOD1 and NOD2
    Article Snippet: .. Subcellular fractionation Membrane and cytosolic fractionation of transfected HEK293 cells was performed using a Subcellular Fractionation Kit (Pierce) as per the manufacturer’s instructions. .. An antibody against GAPDH (Abcam) was used to characterize cytosolic fractions.

    Expressing:

    Article Title: A Genome-Wide Scan for MicroRNA-Related Genetic Variants Associated With Primary Open-Angle Glaucoma
    Article Snippet: .. HEK293 cells (n = 10,000) were plated into 96-well plates and cotransfected with 1 μg pGL3 containing the 3′UTR with either the major or minor allele, miRNA mimic (mirVana Mimics; Thermo Fischer Scientific, Waltham, MA, USA), and a plasmid expressing the Renilla luciferase that served as transfection control, with Lipofectamine RNAiMAX (Invitrogen). .. Luciferase activity was measured with the Dual-Glo Luciferase Assay System according to manufacturer's protocol (Promega).

    Article Title: Toxoplasma gondii cathepsin proteases are undeveloped prominent vaccine antigens against toxoplasmosis
    Article Snippet: .. TgCPB and TgCPL expression in mammalian cells When the cell density reached 80–90%, HEK293 cells were transfected with pBudCE4.1-TgCPB or pBudCE4.1-TgCPL using Lipofectamine™ 2000 reagent (Invitrogen, USA). .. After 24-h incubation, the cells were fixed with cold methanol for 20 min and protein expression was evaluated using an indirect fluorescence antibody test as previously described [ ].

    Article Title: Lamin B is a target for selective nuclear PQC by BAG3: implication for nuclear envelopathies
    Article Snippet: .. Expression of mutant protein was verified in HEK293 cells by Lipofectamine (Thermo Fisher) transfection. .. The plasmids mCherry-lamin B1-10 and mCherry-lamin A-C-18 were a kind gift from Michael Davidson lab (Addgene plasmid #55069), as were HA-p62 from Qing Zhong lab (Addgene plasmid #28027), and GFP-Ub from Nico Dantuma (Addgene #11928).

    Modification:

    Article Title: LRRK2 functions as a Wnt signaling scaffold, bridging cytosolic proteins and membrane-localized LRP6
    Article Snippet: .. HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) plus 100 U/ml penicillin G and 100 μg/ml streptomycin and were transfected using Lipofectamine LTX (Invitrogen) at a 2:1 μl transfection reagent to μg DNA ratio. .. SH-SY5Y cells were cultured in a 1:1 mixture of DMEM and F12 Ham's media supplemented with 10% FBS plus penicillin and streptomycin and were transfected using FuGENE 6 (Roche) at a 2.5:1 μl transfection reagent to μg DNA ratio.

    Plasmid Preparation:

    Article Title: A Genome-Wide Scan for MicroRNA-Related Genetic Variants Associated With Primary Open-Angle Glaucoma
    Article Snippet: .. HEK293 cells (n = 10,000) were plated into 96-well plates and cotransfected with 1 μg pGL3 containing the 3′UTR with either the major or minor allele, miRNA mimic (mirVana Mimics; Thermo Fischer Scientific, Waltham, MA, USA), and a plasmid expressing the Renilla luciferase that served as transfection control, with Lipofectamine RNAiMAX (Invitrogen). .. Luciferase activity was measured with the Dual-Glo Luciferase Assay System according to manufacturer's protocol (Promega).

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    Thermo Fisher hek293 cells
    The impact of SNP rs2273626 in the seed sequence of miR-4707 on miRNA production and targeting. (A) The figure shows the predicted hairpin structure of miR-4707 containing rs2273626, which was associated with VCDR and cup area. The mature miRNA sequences (3p and 5p) are shown in red and the position of variants is depicted by an arrow. To examine the effect of rs2273626 on the miR-4707 expression level, <t>HEK293</t> cells were transfected with GFP-miRNA transcripts containing either the minor allele T or the major allele G. The levels of mature miRNA relative to GFP transcript levels were calculated. (B) Luciferase reporter assays indicating miR-4707-3p–mediated repression of CARD10. HEK293 cells were cotransfected with CARD10 3′UTR luciferase reporter vector and GFP-miRNA transcripts containing either the minor allele T or the major allele G. This experiment indicates a significant difference (P = 0.04) between the relative luciferase activity of the CARD10 3′UTR construct in the presence of miR-4707-3p containing the major allele and the minor allele. Our results suggest that rs2273626 diminishes the regulatory interaction between miR-4707-3p and CARD10, resulting in increased CARD10 levels. All experiments were performed in triplicates and repeated at least three times. Error bars represent standard deviation (SD). NS, nonsignificant.
    Hek293 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 402 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Co-immunoprecipitations. ( A ) <t>Immunoprecipitation</t> and input of SK2-S plus SK2-L. Western blot probed with anti-SK2 antibody raised in rabbit. First three lanes show immunoprecipitates from transfected <t>HEK293</t> cell lysates co-transfected with empty vector (mock), C8-MPP2, or PSD-95 (see Figure 2 ). Immunoprecipitations were performed using anti-SK2 antibody raised in guinea pig. Last three lanes show input material prior to immunoprecipitation. ( B ) Co-immunoprecipitation of C8-SAP-97 co-expressed in HEK293 cells with GluA1 but not with myc-SK2-S. Western blot using anti-C8 antibody detects input of C8-SAP-97 co-expressed with myc-SK2-S or GluA1. C8-SAP-97 co-immunoprecipitated with anti-GluA1 antibody but not with anti-SK2 antibody or IgG. ( C ) Western blot using anti-myc antibody detects myc-SK2-S co-expressed with C8-SAP-97, input and after immunprecipitation with anti-SK2 antibody. Higher MW bands correspond to aggregates of SK2-S. ( D ) Western blot using mouse monoclonal anti-GluA1 antibody detects GluA1 co-expressed with C8-SAP-97, for GluA1 input and GluA1 immunoprecipitated with rabbit polyclonal anti-GluA1 antibody. DOI: http://dx.doi.org/10.7554/eLife.12637.007
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    Thermo Fisher human hek293 cells
    Comparative analysis of miR-15/107 family members in vitro . ( a ) Mature miRNA sequences are shown. Seed sequences are shown in red. The corresponding binding sites within the amyloid precursor protein (APP) and BACE1 3'UTRs are shown in gray. ( b ) Schematic representation (not to scale) of the luciferase reporter construct. Luc, luciferase gene; TK, thymidine kinase promoter. ( c ) APP 3'UTR regulation by selected miR-15/107 family members. <t>HEK293</t> cells were transfected with 50 nmol/l final concentration of candidate mimics. Twenty-four hours post-transfection luciferase signal was measured. Signals were normalized for transfection efficiency, and graph represents the relative luciferase signals compared to the scrambled control (SCR). Statistical significance was assessed by one-way analysis of variance (ANOVA) with Bonferroni multiple comparison test. * P
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    The impact of SNP rs2273626 in the seed sequence of miR-4707 on miRNA production and targeting. (A) The figure shows the predicted hairpin structure of miR-4707 containing rs2273626, which was associated with VCDR and cup area. The mature miRNA sequences (3p and 5p) are shown in red and the position of variants is depicted by an arrow. To examine the effect of rs2273626 on the miR-4707 expression level, HEK293 cells were transfected with GFP-miRNA transcripts containing either the minor allele T or the major allele G. The levels of mature miRNA relative to GFP transcript levels were calculated. (B) Luciferase reporter assays indicating miR-4707-3p–mediated repression of CARD10. HEK293 cells were cotransfected with CARD10 3′UTR luciferase reporter vector and GFP-miRNA transcripts containing either the minor allele T or the major allele G. This experiment indicates a significant difference (P = 0.04) between the relative luciferase activity of the CARD10 3′UTR construct in the presence of miR-4707-3p containing the major allele and the minor allele. Our results suggest that rs2273626 diminishes the regulatory interaction between miR-4707-3p and CARD10, resulting in increased CARD10 levels. All experiments were performed in triplicates and repeated at least three times. Error bars represent standard deviation (SD). NS, nonsignificant.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: A Genome-Wide Scan for MicroRNA-Related Genetic Variants Associated With Primary Open-Angle Glaucoma

    doi: 10.1167/iovs.17-22410

    Figure Lengend Snippet: The impact of SNP rs2273626 in the seed sequence of miR-4707 on miRNA production and targeting. (A) The figure shows the predicted hairpin structure of miR-4707 containing rs2273626, which was associated with VCDR and cup area. The mature miRNA sequences (3p and 5p) are shown in red and the position of variants is depicted by an arrow. To examine the effect of rs2273626 on the miR-4707 expression level, HEK293 cells were transfected with GFP-miRNA transcripts containing either the minor allele T or the major allele G. The levels of mature miRNA relative to GFP transcript levels were calculated. (B) Luciferase reporter assays indicating miR-4707-3p–mediated repression of CARD10. HEK293 cells were cotransfected with CARD10 3′UTR luciferase reporter vector and GFP-miRNA transcripts containing either the minor allele T or the major allele G. This experiment indicates a significant difference (P = 0.04) between the relative luciferase activity of the CARD10 3′UTR construct in the presence of miR-4707-3p containing the major allele and the minor allele. Our results suggest that rs2273626 diminishes the regulatory interaction between miR-4707-3p and CARD10, resulting in increased CARD10 levels. All experiments were performed in triplicates and repeated at least three times. Error bars represent standard deviation (SD). NS, nonsignificant.

    Article Snippet: HEK293 cells (n = 10,000) were plated into 96-well plates and cotransfected with 1 μg pGL3 containing the 3′UTR with either the major or minor allele, miRNA mimic (mirVana Mimics; Thermo Fischer Scientific, Waltham, MA, USA), and a plasmid expressing the Renilla luciferase that served as transfection control, with Lipofectamine RNAiMAX (Invitrogen).

    Techniques: Sequencing, Expressing, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Construct, Standard Deviation

    BAG3 interacts with nuclear protein lamin B using BAG domain. a Co-immunoprecipitation studies show that HSP70 co-immunoprecipitated with BAG3. HEK293 cells were transfected with BAG3 plasmid for 48 h and immunoprecipitation was done with FLAG-tag antibody. Western blot was done with HSP70 antibody. b , c Immunoprecipitation study shows that BAG3 can interact with nuclear envelop protein lamin B but not with lamin A/C. HEK293 cells were co-transfected with BAG3 and lamin B-mCherry or BAG3 and lamin A/C-mCherry. Immunoprecipitation was done with the FLAG antibody and western blots were done using lamin B or lamin A/C antibody, respectively. d Western blot shows that BAG3 interacts with the ubiquitin. HEK293 cells were co-transfected with BAG3 and ubiquitin expressor plasmids. Immunoprecipitation was done with FLAG antibody and western blots were done with ubiquitin antibody. e Schematic diagram shows different domains of BAG3. f Western blot analysis showing detection of full-length BAG3 and the BAG3 deletion mutants by anti-FLAG. g–i Co-immunoprecipitation of BAG3 (using anti-FLAG antibody) followed by western blot for detection of lamin B as well as the BAG3 partners, HSP70 and DNAJ, respectively. j Representative images show that BAG domain can restore the wild-type phenotype in the CRISPR-mediated BAG3 mutant C2C12 cells. Wild-type and mutant C2C12 cells were transfected with the mutant and wild-type BAG3 gene using Lipofectamine for 48 h. Cells were fixed with 4% PFA and immunocytochemistry was done with the FLAG-tag antibody (green) and nuclei were stained with DAPI (blue). Images were captured with confocal microscopy. k Quantification of the results (shown in j ) in which nuclear area was measured using the ImageJ software ( n = 100 cells, in each group)

    Journal: Cell Death & Disease

    Article Title: Lamin B is a target for selective nuclear PQC by BAG3: implication for nuclear envelopathies

    doi: 10.1038/s41419-018-1255-9

    Figure Lengend Snippet: BAG3 interacts with nuclear protein lamin B using BAG domain. a Co-immunoprecipitation studies show that HSP70 co-immunoprecipitated with BAG3. HEK293 cells were transfected with BAG3 plasmid for 48 h and immunoprecipitation was done with FLAG-tag antibody. Western blot was done with HSP70 antibody. b , c Immunoprecipitation study shows that BAG3 can interact with nuclear envelop protein lamin B but not with lamin A/C. HEK293 cells were co-transfected with BAG3 and lamin B-mCherry or BAG3 and lamin A/C-mCherry. Immunoprecipitation was done with the FLAG antibody and western blots were done using lamin B or lamin A/C antibody, respectively. d Western blot shows that BAG3 interacts with the ubiquitin. HEK293 cells were co-transfected with BAG3 and ubiquitin expressor plasmids. Immunoprecipitation was done with FLAG antibody and western blots were done with ubiquitin antibody. e Schematic diagram shows different domains of BAG3. f Western blot analysis showing detection of full-length BAG3 and the BAG3 deletion mutants by anti-FLAG. g–i Co-immunoprecipitation of BAG3 (using anti-FLAG antibody) followed by western blot for detection of lamin B as well as the BAG3 partners, HSP70 and DNAJ, respectively. j Representative images show that BAG domain can restore the wild-type phenotype in the CRISPR-mediated BAG3 mutant C2C12 cells. Wild-type and mutant C2C12 cells were transfected with the mutant and wild-type BAG3 gene using Lipofectamine for 48 h. Cells were fixed with 4% PFA and immunocytochemistry was done with the FLAG-tag antibody (green) and nuclei were stained with DAPI (blue). Images were captured with confocal microscopy. k Quantification of the results (shown in j ) in which nuclear area was measured using the ImageJ software ( n = 100 cells, in each group)

    Article Snippet: Expression of mutant protein was verified in HEK293 cells by Lipofectamine (Thermo Fisher) transfection.

    Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, FLAG-tag, Western Blot, CRISPR, Mutagenesis, Immunocytochemistry, Staining, Confocal Microscopy, Software

    Co-immunoprecipitations. ( A ) Immunoprecipitation and input of SK2-S plus SK2-L. Western blot probed with anti-SK2 antibody raised in rabbit. First three lanes show immunoprecipitates from transfected HEK293 cell lysates co-transfected with empty vector (mock), C8-MPP2, or PSD-95 (see Figure 2 ). Immunoprecipitations were performed using anti-SK2 antibody raised in guinea pig. Last three lanes show input material prior to immunoprecipitation. ( B ) Co-immunoprecipitation of C8-SAP-97 co-expressed in HEK293 cells with GluA1 but not with myc-SK2-S. Western blot using anti-C8 antibody detects input of C8-SAP-97 co-expressed with myc-SK2-S or GluA1. C8-SAP-97 co-immunoprecipitated with anti-GluA1 antibody but not with anti-SK2 antibody or IgG. ( C ) Western blot using anti-myc antibody detects myc-SK2-S co-expressed with C8-SAP-97, input and after immunprecipitation with anti-SK2 antibody. Higher MW bands correspond to aggregates of SK2-S. ( D ) Western blot using mouse monoclonal anti-GluA1 antibody detects GluA1 co-expressed with C8-SAP-97, for GluA1 input and GluA1 immunoprecipitated with rabbit polyclonal anti-GluA1 antibody. DOI: http://dx.doi.org/10.7554/eLife.12637.007

    Journal: eLife

    Article Title: Membrane palmitoylated protein 2 is a synaptic scaffold protein required for synaptic SK2-containing channel function

    doi: 10.7554/eLife.12637

    Figure Lengend Snippet: Co-immunoprecipitations. ( A ) Immunoprecipitation and input of SK2-S plus SK2-L. Western blot probed with anti-SK2 antibody raised in rabbit. First three lanes show immunoprecipitates from transfected HEK293 cell lysates co-transfected with empty vector (mock), C8-MPP2, or PSD-95 (see Figure 2 ). Immunoprecipitations were performed using anti-SK2 antibody raised in guinea pig. Last three lanes show input material prior to immunoprecipitation. ( B ) Co-immunoprecipitation of C8-SAP-97 co-expressed in HEK293 cells with GluA1 but not with myc-SK2-S. Western blot using anti-C8 antibody detects input of C8-SAP-97 co-expressed with myc-SK2-S or GluA1. C8-SAP-97 co-immunoprecipitated with anti-GluA1 antibody but not with anti-SK2 antibody or IgG. ( C ) Western blot using anti-myc antibody detects myc-SK2-S co-expressed with C8-SAP-97, input and after immunprecipitation with anti-SK2 antibody. Higher MW bands correspond to aggregates of SK2-S. ( D ) Western blot using mouse monoclonal anti-GluA1 antibody detects GluA1 co-expressed with C8-SAP-97, for GluA1 input and GluA1 immunoprecipitated with rabbit polyclonal anti-GluA1 antibody. DOI: http://dx.doi.org/10.7554/eLife.12637.007

    Article Snippet: Co-immunoprecipitation HEK293 cells were transfected using Lipofectamine 2000 (Thermo Scientific).

    Techniques: Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation

    Comparative analysis of miR-15/107 family members in vitro . ( a ) Mature miRNA sequences are shown. Seed sequences are shown in red. The corresponding binding sites within the amyloid precursor protein (APP) and BACE1 3'UTRs are shown in gray. ( b ) Schematic representation (not to scale) of the luciferase reporter construct. Luc, luciferase gene; TK, thymidine kinase promoter. ( c ) APP 3'UTR regulation by selected miR-15/107 family members. HEK293 cells were transfected with 50 nmol/l final concentration of candidate mimics. Twenty-four hours post-transfection luciferase signal was measured. Signals were normalized for transfection efficiency, and graph represents the relative luciferase signals compared to the scrambled control (SCR). Statistical significance was assessed by one-way analysis of variance (ANOVA) with Bonferroni multiple comparison test. * P

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Preclinical Evaluation of miR-15/107 Family Members as Multifactorial Drug Targets for Alzheimer's Disease

    doi: 10.1038/mtna.2015.33

    Figure Lengend Snippet: Comparative analysis of miR-15/107 family members in vitro . ( a ) Mature miRNA sequences are shown. Seed sequences are shown in red. The corresponding binding sites within the amyloid precursor protein (APP) and BACE1 3'UTRs are shown in gray. ( b ) Schematic representation (not to scale) of the luciferase reporter construct. Luc, luciferase gene; TK, thymidine kinase promoter. ( c ) APP 3'UTR regulation by selected miR-15/107 family members. HEK293 cells were transfected with 50 nmol/l final concentration of candidate mimics. Twenty-four hours post-transfection luciferase signal was measured. Signals were normalized for transfection efficiency, and graph represents the relative luciferase signals compared to the scrambled control (SCR). Statistical significance was assessed by one-way analysis of variance (ANOVA) with Bonferroni multiple comparison test. * P

    Article Snippet: Mouse neuroblastoma Neuro2a cells, mouse Neuro2a cells expressing the Swedish mutant of APP and Δ9 mutant of PSEN1 (Neuro2a APPSwe/Δ9) (Dr. Gopal Thinakaran, University of Chicago, USA), mouse hippocampal-derived HT22 cells (Dr. Schubert, Salk institute, USA), human HEK293T cells, and human HEK293 cells expressing the Swedish mutant of APP (HEK293-APPSwe) were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum (ThermoFischer Scientific, Waltham, MA).

    Techniques: In Vitro, Binding Assay, Luciferase, Construct, Transfection, Concentration Assay