primary antibodies include rabbit anti eag1  (Alomone Labs)

Journal: Cell & Bioscience

Article Title: 14-3-3 proteins regulate cullin 7-mediated Eag1 degradation

doi: 10.1186/s13578-023-00969-w

Figure Lengend Snippet: Difopein increases Eag1 protein expression. A ( Left ) Representative immunoblot showing the effect of difopein co-expression on Eag1 over-expressed in HEK293T cells. Cells were transfected with cDNAs for Eag1, as well as YFP vector, YFP-difopein, or YFP-R18 mutant (YFP-R18 mut). Two days post-transfection, cells were subject to immunoblotting analyses with the anti-Eag1 (α-Eag1), anti-GFP (α-GFP), and anti-β-actin (α-actin) antibodies. ( Right ) Quantitative analyses of relative Eag1 protein levels for the three co-transfection conditions. Protein densities were standardized as the ratio to the cognate β-actin signals, followed by normalization with respect to the YFP vector control (*, P < 0.05; n = 5). B Lack of effect of difopein co-expression on Eag1 mRNA level in HEK293T cells. Semi-quantitative RT-PCR analyses of relative Eag1 mRNA levels were employed for the three co-expression conditions. mRNA levels of Eag1 were standardized as the ratio of Eag1 signals to the cognate GAPDH mRNA levels, followed by normalization with respect to the YFP vector control (n = 3). C–D Effects of shRNA knockdown of various endogenous 14-3-3 proteins on Eag1 protein ( C ) or mRNA ( D ) levels in HEK293T cells. HEK293T cells over-expressing Eag1 were subject to infection with a control shRNA for GFP (sh-GFP), or shRNA specific for 14-3-3β, η, or θ isoforms (sh-14-3-3β#1, sh-14-3-3β#2, sh-14-3-3η, sh-14-3-3θ#1, sh-14-3-3θ#2). Quantitative analyses of relative Eag1 protein levels are based on normalization with respect to the sh-GFP control (*, P < 0.05; n = 4). Lack of effect of 14-3-3 knockdown on Eag1 mRNA levels is supported by quantitative analyses of relative Eag1 mRNA levels normalized with respect to the sh-GFP control (n = 5)

Article Snippet: Primary antibodies include rabbit anti-Eag1 (1:15000; Alomone, Jerusalem, Israel), mouse anti-Flag (1:5000; Sigma, St. Louis, MO, USA), rabbit anti-Flag (1:5000; Sigma, Sigma, St. Louis, MO, USA), mouse anti-Cul7 (1:5000; Sigma, St. Louis, MO, USA), mouse anti-ubiquitin (FK2, 1:1000; Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-GAPDH (1:50000; GeneTex, Irvine, CA, USA), mouse anti-GFP (1:10000; Abcam, Cambridge, MA, USA), mouse anti-β-Actin (1:5000; Sigma, St. Louis, MO, USA), mouse anti-GST (1:5000; Cell signaling Technology, Danvers, MA, USA), and mouse anti-Myc (clone 9E10) antibodies.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Cotransfection, Quantitative RT-PCR, shRNA, Infection

Journal: Cell & Bioscience

Article Title: 14-3-3 proteins regulate cullin 7-mediated Eag1 degradation

doi: 10.1186/s13578-023-00969-w

Figure Lengend Snippet: Difopein promotes Eag1 protein stability. A Difopein co-expression enhances Eag1 protein half-life values in HEK293T cells. ( Upper left ) Representative immunoblots showing the protein turn-over time course of Eag1 co-expressed with R18 mutant or difopein in HEK293T cells. 48 h post-transfection, cells were subject to 100 μg/ml cycloheximide (CHX) treatment for the indicated durations. ( Lower left ) Linear plot of relative Eag1 protein levels in response to different CHX treatment durations. Protein densities were standardized as the ratio of Eag1 signals to the cognate GAPDH signals, followed by normalization with respect to the no-CHX-treatment (0 h) control. ( Lower right ) Semi-logarithmic plot of linear-regression analyses ( solid lines ) of the same data points shown to the left. ( Upper right ) Statistical comparisons of Eag1 protein half-life values for the two co-expression conditions (*, P < 0.05; n = 3). B , C Difopein co-expression diminishes Eag1 protein ubiquitination. Transfected cells were treated with 10 μM MG132 ( B ) or 100 μM chloroquine (CQ) ( C ) for eight hours, followed by immunoprecipitation (IP) with the anti-Eag1 antibody. ( Top panels ) Representative immunoblot showing Eag1 ubiquitination in the absence or presence of difopein co-expression. Ubiquitinated Eag1 is visualized as high-molecular-weight protein smears detected with the FK2 anti-ubiquitin antibody. ( Bottom panels ) Quantification of relative ubiquitinated Eag1 levels normalized with respect to the R18 mutant control (*, P < 0.05; n = 3). D , E Difopein co-expression abolishes MG132- ( D ) and CQ- ( E ) induced increase in Eag1 protein level. ( Left panels ) Representative immunoblots. ( Right panels ) Quantitative analyses of the effect of MG132 ( D ) and CQ ( E ) treatments on Eag1 protein levels for the two co-expression conditions. Protein densities were standardized as the ratio to the cognate GAPDH signals, followed by normalization with respect to the corresponding vehicle-treated control (*, P < 0.05; n = 6)

Article Snippet: Primary antibodies include rabbit anti-Eag1 (1:15000; Alomone, Jerusalem, Israel), mouse anti-Flag (1:5000; Sigma, St. Louis, MO, USA), rabbit anti-Flag (1:5000; Sigma, Sigma, St. Louis, MO, USA), mouse anti-Cul7 (1:5000; Sigma, St. Louis, MO, USA), mouse anti-ubiquitin (FK2, 1:1000; Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-GAPDH (1:50000; GeneTex, Irvine, CA, USA), mouse anti-GFP (1:10000; Abcam, Cambridge, MA, USA), mouse anti-β-Actin (1:5000; Sigma, St. Louis, MO, USA), mouse anti-GST (1:5000; Cell signaling Technology, Danvers, MA, USA), and mouse anti-Myc (clone 9E10) antibodies.

Techniques: Expressing, Western Blot, Mutagenesis, Transfection, Immunoprecipitation, Molecular Weight

Journal: Cell & Bioscience

Article Title: 14-3-3 proteins regulate cullin 7-mediated Eag1 degradation

doi: 10.1186/s13578-023-00969-w

Figure Lengend Snippet: Difopein increases both immature and mature Eag1 protein levels. A Difopein co-expression promotes immature Eag1 protein stability in HEK293T cells. (Left ) Representative immunoblot showing the effect of R18 mutant or difopein co-expression on Eag1 protein turn-over time course in the presence of brefeldin A (BFA). Transfected HEK293T cells were pretreated with BFA (10 μM) for 12 h, followed by cycloheximide (CHX) treatment for the indicated duration. ( Center ) Linear plot of Eag1 protein degradation time course in the presence of BFA treatment. ( Right ) Semi-logarithmic plot of linear-regression analyses ( solid lines ) of the same data points shown to the left. Protein densities were standardized as the ratio of Eag1 signals to the cognate GAPDH signals, followed by normalization with respect to the corresponding no-CHX-treatment (0 h) control. Data points represent the average of four independent experiments. B Difopein co-expression augments cell-surface Eag1 protein level. ( Left panels ) Representative immunoblots. Cell lysates from biotinylated intact cells were either directly employed for immunoblotting analyses ( Total ) or subject to streptavidin pull-down prior to immunoblotting analyses ( Surface ). Actin was used as the loading control. ( Right panels ) Quantification of total and surface protein levels, as well as membrane trafficking efficiency (Surface/total). Total and surface protein densities were standardized as the ratio to the cognate total actin signal, followed by normalization with that of the R18 mutant control. Membrane trafficking efficiency was calculated as surface protein density divided by the corresponding standardized total protein density, followed by normalization with respect to the surface/total ratio of the R18 mutant control (*, P < 0.05; n = 3)

Article Snippet: Primary antibodies include rabbit anti-Eag1 (1:15000; Alomone, Jerusalem, Israel), mouse anti-Flag (1:5000; Sigma, St. Louis, MO, USA), rabbit anti-Flag (1:5000; Sigma, Sigma, St. Louis, MO, USA), mouse anti-Cul7 (1:5000; Sigma, St. Louis, MO, USA), mouse anti-ubiquitin (FK2, 1:1000; Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-GAPDH (1:50000; GeneTex, Irvine, CA, USA), mouse anti-GFP (1:10000; Abcam, Cambridge, MA, USA), mouse anti-β-Actin (1:5000; Sigma, St. Louis, MO, USA), mouse anti-GST (1:5000; Cell signaling Technology, Danvers, MA, USA), and mouse anti-Myc (clone 9E10) antibodies.

Techniques: Expressing, Western Blot, Mutagenesis, Transfection

Journal: Cell & Bioscience

Article Title: 14-3-3 proteins regulate cullin 7-mediated Eag1 degradation

doi: 10.1186/s13578-023-00969-w

Figure Lengend Snippet: Difopein enhances both ER and cell-surface expression of Eag1. Representative confocal micrographs showing the effect of R18 mutant and difopein co-expression on Eag1 immunofluorescent signals in HEK293T cells, in the absence ( A ) or presence of 12-h treatment with 10 μM MG132 ( B ) or 100 μM chloroquine (CQ) ( C ). Eag1 was detected with the anti-Eag1 antibody ( cyan ), nuclei were counterstained with DAPI ( blue ), and YFP-tagged proteins were directly visualized ( green ). ER and cell-surface localizations of Eag1 were determined by co-localization with the ER marker calnexin (red; left panels) and the plasma membrane marker cadherin ( red; right panels ), respectively. Merged images are shown in the third column of each panel. Arrows indicate intracellular ER staining, whereas arrowheads denote plasma membrane staining. Scale bar, 10 μm. Data shown here are representative of over 80 cells from at least three independent experiments. See Additional file : Fig. S3 for further quantitative analyses

Article Snippet: Primary antibodies include rabbit anti-Eag1 (1:15000; Alomone, Jerusalem, Israel), mouse anti-Flag (1:5000; Sigma, St. Louis, MO, USA), rabbit anti-Flag (1:5000; Sigma, Sigma, St. Louis, MO, USA), mouse anti-Cul7 (1:5000; Sigma, St. Louis, MO, USA), mouse anti-ubiquitin (FK2, 1:1000; Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-GAPDH (1:50000; GeneTex, Irvine, CA, USA), mouse anti-GFP (1:10000; Abcam, Cambridge, MA, USA), mouse anti-β-Actin (1:5000; Sigma, St. Louis, MO, USA), mouse anti-GST (1:5000; Cell signaling Technology, Danvers, MA, USA), and mouse anti-Myc (clone 9E10) antibodies.

Techniques: Expressing, Mutagenesis, Marker, Staining

Journal: Cell & Bioscience

Article Title: 14-3-3 proteins regulate cullin 7-mediated Eag1 degradation

doi: 10.1186/s13578-023-00969-w

Figure Lengend Snippet: Difopein up-regulates Eag1 protein expression in neurons. A , B Over-expression of difopein promotes endogenous Eag1 expression in cultured cortical neurons. Neurons (DIV10) were transfected with YFP, YFP-difopein, or YFP-R18 mutant, and then incubated for two days, followed by immunoblotting ( A ) or immunofluorescent ( B ) analyses. Quantitative analyses of immunoblots are summarized by the bar graphs. Protein densities were standardized as the ratio to the cognate GAPDH signals, followed by normalization with respect to the YFP vector control (*, P < 0.05; n = 5). Endogenous Eag1 was detected with the anti-Eag1 antibody ( red ), and YFP-tagged proteins were directly visualized ( green ). Arrowheads denote punctate Eag1 staining patterns. Scale bar, 10 μm. See Additional file : Fig. S4 for further quantitative analyses of immunofluorescent images. C shRNA knockdown of 14-3-3β and 14-3-3θ, but not 14-3-3η, increases endogenous Eag1 protein level in cultured cortical neurons. Neurons (DIV10) were infected with various viral stocks and selected with puromycin; two days post-infection, immunoblotting analyses were performed using the indicated antibodies

Article Snippet: Primary antibodies include rabbit anti-Eag1 (1:15000; Alomone, Jerusalem, Israel), mouse anti-Flag (1:5000; Sigma, St. Louis, MO, USA), rabbit anti-Flag (1:5000; Sigma, Sigma, St. Louis, MO, USA), mouse anti-Cul7 (1:5000; Sigma, St. Louis, MO, USA), mouse anti-ubiquitin (FK2, 1:1000; Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-GAPDH (1:50000; GeneTex, Irvine, CA, USA), mouse anti-GFP (1:10000; Abcam, Cambridge, MA, USA), mouse anti-β-Actin (1:5000; Sigma, St. Louis, MO, USA), mouse anti-GST (1:5000; Cell signaling Technology, Danvers, MA, USA), and mouse anti-Myc (clone 9E10) antibodies.

Techniques: Expressing, Over Expression, Cell Culture, Transfection, Mutagenesis, Incubation, Western Blot, Plasmid Preparation, Staining, shRNA, Infection

Journal: Cell & Bioscience

Article Title: 14-3-3 proteins regulate cullin 7-mediated Eag1 degradation

doi: 10.1186/s13578-023-00969-w

Figure Lengend Snippet: Effects of difopein expression on NMDA-induced neuronal excitotoxicity. A , B Difopein rescues NMDA excitotoxicity and averts NMDA-mediated reduction of endogenous Eag1 protein level in cultured cortical neurons. Neurons (DIV10) were subject to transfection with either R18 mutant or difopein. Two days post-transfection, neurons were stimulated with 20 μM NMDA for six hours, in the absence or presence of 30-min pretreatment with the NMDA receptor antagonist AP5 (50 μM), followed by MTT assays ( A ) or immunoblotting analyses with the indicated antibodies ( B ). Cell viability is expressed as the relative optical density at 540 nm of the mitochondria-produced formazan with respect to the non-NMDA-treatment (Untreated) control of YFP-R18-mut-transfected neurons. Statistical comparisons were performed with respect to the untreated group of R18 mutant-transfected neurons (*, P < 0.05; n = 9). C Proteasome inhibition prevents NMDA-mediated reduction of endogenous Eag1 protein level in cultured cortical neurons. Neurons (DIV12) were pretreated with the NMDA receptor antagonist AP5 (50 μM), the proteasomal inhibitors ALLN (10 μM) and MG132 (20 μM), or the caspase inhibitor zVAD-FMK (20 μM) for 30 min. Cells were then treated with 20 μM NMDA for 12 h in the presence of the specified inhibitors, followed by immunoblotting analyses with the indicated antibodies

Article Snippet: Primary antibodies include rabbit anti-Eag1 (1:15000; Alomone, Jerusalem, Israel), mouse anti-Flag (1:5000; Sigma, St. Louis, MO, USA), rabbit anti-Flag (1:5000; Sigma, Sigma, St. Louis, MO, USA), mouse anti-Cul7 (1:5000; Sigma, St. Louis, MO, USA), mouse anti-ubiquitin (FK2, 1:1000; Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-GAPDH (1:50000; GeneTex, Irvine, CA, USA), mouse anti-GFP (1:10000; Abcam, Cambridge, MA, USA), mouse anti-β-Actin (1:5000; Sigma, St. Louis, MO, USA), mouse anti-GST (1:5000; Cell signaling Technology, Danvers, MA, USA), and mouse anti-Myc (clone 9E10) antibodies.

Techniques: Expressing, Cell Culture, Transfection, Mutagenesis, Western Blot, Produced, Inhibition

Journal: Cell & Bioscience

Article Title: 14-3-3 proteins regulate cullin 7-mediated Eag1 degradation

doi: 10.1186/s13578-023-00969-w

Figure Lengend Snippet: Effects of BV02 treatment on NMDA-induced neuronal excitotoxicity. A Treatment with the small-molecule 14-3-3 inhibitor BV02 (40 μM) promotes endogenous Eag1 protein level in cultured cortical neurons. Neurons (DIV12) were pretreated with the indicated concentrations of BV02 for six hours, followed by immunoblotting analyses with the indicated antibodies. B – D BV02 (40 μM) treatment averts NMDA-mediated reduction of endogenous Eag1 protein level and rescues NMDA excitotoxicity in cultured cortical neurons. Neurons were pretreated with DMSO or BV02, and then subject to 6-h treatment of 20 μM NMDA (in the absence or presence of 30-min 50-μM AP5 pretreatment), followed by immunoblotting analyses ( B ), MTT assay ( C ), or immunofluorescent inspections ( D ). Cell viability is expressed as the relative optical density at 540 nm of formazan with respect to the non-NMDA-treatment (Untreated) control of DMSO-treated neurons. Statistical comparisons were performed with respect to the untreated group of DMSO-treated neurons (*, P < 0.05; n = 5). For immunofluorescent experiments, neurons were stained with the anti-MAP2 antibody ( red ) and the nucleus counterstain DAPI ( blue ). NMDA treatment led to a significant diminishment of immunofluorescent signals of MAP2 (but not those of DAPI), which was prevented by pretreatment with AP5 or BV02. Scale bar, 10 μm

Article Snippet: Primary antibodies include rabbit anti-Eag1 (1:15000; Alomone, Jerusalem, Israel), mouse anti-Flag (1:5000; Sigma, St. Louis, MO, USA), rabbit anti-Flag (1:5000; Sigma, Sigma, St. Louis, MO, USA), mouse anti-Cul7 (1:5000; Sigma, St. Louis, MO, USA), mouse anti-ubiquitin (FK2, 1:1000; Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-GAPDH (1:50000; GeneTex, Irvine, CA, USA), mouse anti-GFP (1:10000; Abcam, Cambridge, MA, USA), mouse anti-β-Actin (1:5000; Sigma, St. Louis, MO, USA), mouse anti-GST (1:5000; Cell signaling Technology, Danvers, MA, USA), and mouse anti-Myc (clone 9E10) antibodies.

Techniques: Cell Culture, Western Blot, MTT Assay, Staining

Journal: Cell & Bioscience

Article Title: 14-3-3 proteins regulate cullin 7-mediated Eag1 degradation

doi: 10.1186/s13578-023-00969-w

Figure Lengend Snippet: Difopein reduces Eag1 degradation by Cul7. A The effect of R18 mutant or difopein on Eag1 degradation by Myc-tagged Cul7 (Myc-Cul7) in HEK293T cells. (Left ) Representative immunoblots. Eag1 was co-expressed with increasing amounts of Cul7. ( Right ) Quantification of relative Eag1 protein levels with respect to the amount of Cul7 used for co-transfection. Protein densities were standardized as the ratio of Eag1 signals to the cognate GAPDH signals, followed by normalization with respect to the Myc-vector control (n = 3). B The effect of siRNA knock-down of endogenous Cul7 (siCul7) on the regulation of Eag1 protein expression by R18 mutant or difopein in HEK293T cells. (Left ) Representative immunoblots. ( Right ) Quantification of relative Eag1 protein levels. Protein densities were standardized as the ratio of Eag1 signals to the cognate β-actin signals, followed by normalization with respect to the corresponding siRNA negative control (siControl) or R18 mutant control (*, p < 0.05; n = 3)

Article Snippet: Primary antibodies include rabbit anti-Eag1 (1:15000; Alomone, Jerusalem, Israel), mouse anti-Flag (1:5000; Sigma, St. Louis, MO, USA), rabbit anti-Flag (1:5000; Sigma, Sigma, St. Louis, MO, USA), mouse anti-Cul7 (1:5000; Sigma, St. Louis, MO, USA), mouse anti-ubiquitin (FK2, 1:1000; Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-GAPDH (1:50000; GeneTex, Irvine, CA, USA), mouse anti-GFP (1:10000; Abcam, Cambridge, MA, USA), mouse anti-β-Actin (1:5000; Sigma, St. Louis, MO, USA), mouse anti-GST (1:5000; Cell signaling Technology, Danvers, MA, USA), and mouse anti-Myc (clone 9E10) antibodies.

Techniques: Mutagenesis, Western Blot, Cotransfection, Plasmid Preparation, Expressing, Negative Control

Journal: Cell & Bioscience

Article Title: 14-3-3 proteins regulate cullin 7-mediated Eag1 degradation

doi: 10.1186/s13578-023-00969-w

Figure Lengend Snippet: Difopein disrupts Eag1 interaction with Cul7. A GST pull-down assay of the interaction of Cul7 with Eag1 C-terminal region. Shown on the top is the structural topology for Eag1, as well as two GST-Eag1 C-terminal fusion proteins, GST-Eag1-C1-A and GST-Eag1-C1-B. ( Left ) Cell lysates prepared from HEK293T cells expressing Myc-Cul7 were used for pull-down assay with GST, GST-Eag1-C1-A, or GST-Eag1-C1-B, followed by immunoblotting with the anti-Cul7 or anti-GST antibodies. ( Center ) Myc-Cul7 was co-expressed with either YFP-R18 mutant or YFP-difopein in HEK293T cells, followed by pull-down assay with GST-Eag1-C1-B. ( Right ) Quantification of the relative pull-down efficiency. Protein densities were standardized as the ratio of Cul7 pull-down signals to the corresponding input signals, followed by normalization with respect to the YFP-R18 mutant co-expression control (*, p < 0.05; n = 3). B GST pull-down assay of the interaction of Cul7 with Eag1 N-terminal region. Shown on the top is the structural topology for the N-terminal fusion protein GST-Eag1-N. ( Left ) Cell lysates prepared from HEK293T cells expressing Myc-Cul7 were used for GST pull-down assay with GST or GST-Eag1-N. ( Center ) Myc-Cul7 was pulled down with GST-Eag1-N in the presence of either YFP-R18 mutant or YFP-difopein. ( Rght ) Quantification of the relative pull-down efficiency (*, p < 0.05; n = 3). C The effect of difopein on the co-immunoprecipitation efficiency of Cul7 and Eag1 in HEK293T cells. ( Left ) Representative immunoblots. Myc-Cul7, Eag1, and YFP-R18 mutant/YFP-difopein were co-expressed in HEK293T cells. 24 h after transfection, cells were treated with 10 μM MG132 for 12 h. Cell lysates were immunoprecipitated ( IP ) with the anti-Cul7 antibody, followed by immunoblotting analyses. ( Right ) Quantification of relative co-immunoprecipitation efficiency of Cul7 and Eag1. Protein densities were standardized as the ratio of Eag1 IP signals to the corresponding input signals, followed by normalization with respect to the YFP-R18 mutant co-expression control (*, p < 0.05; n = 3)

Article Snippet: Primary antibodies include rabbit anti-Eag1 (1:15000; Alomone, Jerusalem, Israel), mouse anti-Flag (1:5000; Sigma, St. Louis, MO, USA), rabbit anti-Flag (1:5000; Sigma, Sigma, St. Louis, MO, USA), mouse anti-Cul7 (1:5000; Sigma, St. Louis, MO, USA), mouse anti-ubiquitin (FK2, 1:1000; Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-GAPDH (1:50000; GeneTex, Irvine, CA, USA), mouse anti-GFP (1:10000; Abcam, Cambridge, MA, USA), mouse anti-β-Actin (1:5000; Sigma, St. Louis, MO, USA), mouse anti-GST (1:5000; Cell signaling Technology, Danvers, MA, USA), and mouse anti-Myc (clone 9E10) antibodies.

Techniques: Pull Down Assay, Expressing, Western Blot, Mutagenesis, Immunoprecipitation, Transfection

Journal: Cell & Bioscience

Article Title: 14-3-3 proteins regulate cullin 7-mediated Eag1 degradation

doi: 10.1186/s13578-023-00969-w

Figure Lengend Snippet: The CNBHD and PAS domain are essential for Eag1 regulation by difopein. A Structural topology for Eag1, hErg, and various Eag1 chimeric constructs. Chimera A: Eag1 containing hErg C-linker. Chimera B: Eag1 containing hErg CNBHD. Chimera C: Eag1 containing hErg post-CNBHD region. Chimera N: Eag1 containing the complete hErg N-terminal region. Chimera P: Eag1 containing hErg PAS domain. Chimera O: Eag1 containing hErg N-linker. B , C Replacement with hErg CNBHD (chimera B), PAS domain (chimeras N and P), or N-linker (chimeras N and O) abolishes the effect of difopein on Eag1 protein level. ( Left panels ) Representative immunoblots. ( Right panels ) Quantification of relative Eag1 protein levels. Myc-tagged Eag1 wild-type (WT) and chimeric constructs were co-transfected with YFP vector, YFP-R18 mutant or YFP-difopein in HEK293T cells. Protein densities were standardized as the ratio of Eag1 signals to the cognate β-actin signals, followed by normalization with respect to the YFP vector control (*, P < 0.05; n = 3–6)

Article Snippet: Primary antibodies include rabbit anti-Eag1 (1:15000; Alomone, Jerusalem, Israel), mouse anti-Flag (1:5000; Sigma, St. Louis, MO, USA), rabbit anti-Flag (1:5000; Sigma, Sigma, St. Louis, MO, USA), mouse anti-Cul7 (1:5000; Sigma, St. Louis, MO, USA), mouse anti-ubiquitin (FK2, 1:1000; Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-GAPDH (1:50000; GeneTex, Irvine, CA, USA), mouse anti-GFP (1:10000; Abcam, Cambridge, MA, USA), mouse anti-β-Actin (1:5000; Sigma, St. Louis, MO, USA), mouse anti-GST (1:5000; Cell signaling Technology, Danvers, MA, USA), and mouse anti-Myc (clone 9E10) antibodies.

Techniques: Construct, Western Blot, Transfection, Plasmid Preparation, Mutagenesis

Journal: Cell & Bioscience

Article Title: 14-3-3 proteins regulate cullin 7-mediated Eag1 degradation

doi: 10.1186/s13578-023-00969-w

Figure Lengend Snippet: 14-3-3 proteins contribute to Cul7-mediated degradation of disease-associated mutant Eag1 proteins. The effect of siRNA knockdown of endogenous Cul7 ( A ), difopein co-expression ( B ), or BV02 treatment ( C ) on WT and mutant Eag1 protein levels in HEK293T cells. ( Left panels ) Representative immunoblots. ( Right panels ) Quantification of relative Eag1 protein levels. Protein densities were standardized as the ratio of Eag1 signals to the cognate β-actin signals, followed by normalization with respect to the YFP-R18 mutant (*, P < 0.05; n = 3)

Article Snippet: Primary antibodies include rabbit anti-Eag1 (1:15000; Alomone, Jerusalem, Israel), mouse anti-Flag (1:5000; Sigma, St. Louis, MO, USA), rabbit anti-Flag (1:5000; Sigma, Sigma, St. Louis, MO, USA), mouse anti-Cul7 (1:5000; Sigma, St. Louis, MO, USA), mouse anti-ubiquitin (FK2, 1:1000; Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-GAPDH (1:50000; GeneTex, Irvine, CA, USA), mouse anti-GFP (1:10000; Abcam, Cambridge, MA, USA), mouse anti-β-Actin (1:5000; Sigma, St. Louis, MO, USA), mouse anti-GST (1:5000; Cell signaling Technology, Danvers, MA, USA), and mouse anti-Myc (clone 9E10) antibodies.

Techniques: Mutagenesis, Expressing, Western Blot

Journal: Cell & Bioscience

Article Title: 14-3-3 proteins regulate cullin 7-mediated Eag1 degradation

doi: 10.1186/s13578-023-00969-w

Figure Lengend Snippet: Protein docking models for the 14-3-3-Eag1-Cul7 complex. Homology modeling of the Cul7 protein complex was based on the structures of cullin 1 (PDB: 1LDK), Skp1-Fbw7 complex (PDB: 2OVP), E2 ubiquitin-conjugating enzyme (PDB: 3CEG), Doc1 homology domain (PDB: 1GQP), and CPH domain (PDB: JNG). A Interaction of 14-3-3θ homodimer ( aqua , green ) with the Cul7 ( tangerine )-Skp1 ( orange )-Fbw8 ( saffron )-Rbx1 ( olive )-E2 ( pumpkin ) protein complex. One 14-3-3θ subunit ( green ) may directly contact a Cul7 loop region between the DOC domain and C-terminal domain, whereas the other 14-3-3θ subunit (aqua) is modeled as a binding partner of the adaptor protein Skp1. B Ternary organization of 14-3-3θ homodimer, Cul7 protein complex, and the PAS domain/CNBHD from three Eag1 subunits ( violet , burgundy , blue ). The Cul7 complex appears to exclusively bind to a single Eag1 subunit ( burgundy ), with the Cul7 C-terminal domain sitting on the surface of the Eag1 PAS domain, and the substrate-targeting subunit Fbw8 directly contacting the Eag1 CNBHD. As in Fig. 13, 14-3-3θ homodimer ( aqua , green ) interacts with the N-linker ( violet )/CNBHD ( burgundy )/post-CNBHD ( blue ) from three distinct Eag1 subunits, respectively. Also shown are two sets of intersubunit PAS domain-CNBHD interaction between neighboring Eag1 subunits ( violet - burgundy ; burgundy - blue ). C Transmembrane, extracellular, and intracellular views of four Cul7 protein complexes, four 14-3-3θ homodimers, and the Eag1 tetramer ( violet , burgundy , blue , salmon ) at the plasma membrane. The docking models in ( A ) and ( B ) are equivalent to the enlargement of a portion of the transmembrane and intracellular views, respectively

Article Snippet: Primary antibodies include rabbit anti-Eag1 (1:15000; Alomone, Jerusalem, Israel), mouse anti-Flag (1:5000; Sigma, St. Louis, MO, USA), rabbit anti-Flag (1:5000; Sigma, Sigma, St. Louis, MO, USA), mouse anti-Cul7 (1:5000; Sigma, St. Louis, MO, USA), mouse anti-ubiquitin (FK2, 1:1000; Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-GAPDH (1:50000; GeneTex, Irvine, CA, USA), mouse anti-GFP (1:10000; Abcam, Cambridge, MA, USA), mouse anti-β-Actin (1:5000; Sigma, St. Louis, MO, USA), mouse anti-GST (1:5000; Cell signaling Technology, Danvers, MA, USA), and mouse anti-Myc (clone 9E10) antibodies.

Techniques: Binding Assay

Journal: Cell & Bioscience

Article Title: 14-3-3 proteins regulate cullin 7-mediated Eag1 degradation

doi: 10.1186/s13578-023-00969-w

Figure Lengend Snippet: Molecular modeling of the binding of 14-3-3θ homodimer to Eag1. Protein docking models based on the structures of human 14-3-3θ (PDB: 2BTP) and rat Eag1 (PDB: 5K7L). A Ribbon representation of a single 14-3-3θ homodimer (colored in aqua and green ) interacting with the N-linker region ( raspberry ) and PAS domain ( violet ) of one Eag1 subunit, the proximal CNBHD ( burgundy ) of a second Eag1 subunit, and the proximal post-CNBHD region ( blue ) of a third Eag1 subunit. The PAS domain ( violet ) from one Eag1 subunit directly interacts with the distal end of the CNBHD ( burgundy ) of a neighboring Eag1 subunit, with the intrinsic ligand motif (YNL) emphasized in lime . A portion of the distal segment of the post-CNBHD region ( blue ), which may also be in contact with 14-3-3, is schematically presented as spheres. The two yellow boxes (~ 15 Å × 15 Å) denote the 14-3-3θ-Eag1 binding regions highlighted in ( B ) and ( C ). B Enlarged view of the 14-3-3θ-Eag1 binding region enclosed by the yellow box to the left in ( A ), highlighting that the H4, H5, and H6 helices ( aqua ) of the same 14-3-3θ subunit are in close proximity (~ 3–5 Å) with the N-linker ( raspberry ) of Eag1. Specific residues in 14-3-3θ and Eag1 are labeled in aqua and raspberry , respectively. C Enlarged view of the 14-3-3θ-Eag1 binding region enclosed by the yellow box to the right in ( A ), highlighting that the H1 helix ( aqua ) from one 14-3-3θ subunit and the H3 helix ( green ) from the other 14-3-3θ subunit are in close proximity (~ 3–5 Å) with the proximal CNBHD ( burgundy ) of Eag1. Specific residues in the two 14-3-3θ subunits are labeled in aqua and green , respectively. D Intracellular view of four 14-3-3θ homodimers (all in aqua and green ) in contact with the Eag1 tetramer ( violet , burgundy , blue , salmon ). CNBHDs are located in the center region, directly interacting with PAS domains from neighboring Eag1 subunits

Article Snippet: Primary antibodies include rabbit anti-Eag1 (1:15000; Alomone, Jerusalem, Israel), mouse anti-Flag (1:5000; Sigma, St. Louis, MO, USA), rabbit anti-Flag (1:5000; Sigma, Sigma, St. Louis, MO, USA), mouse anti-Cul7 (1:5000; Sigma, St. Louis, MO, USA), mouse anti-ubiquitin (FK2, 1:1000; Enzo Life Sciences, Farmingdale, NY, USA), rabbit anti-GAPDH (1:50000; GeneTex, Irvine, CA, USA), mouse anti-GFP (1:10000; Abcam, Cambridge, MA, USA), mouse anti-β-Actin (1:5000; Sigma, St. Louis, MO, USA), mouse anti-GST (1:5000; Cell signaling Technology, Danvers, MA, USA), and mouse anti-Myc (clone 9E10) antibodies.

Techniques: Binding Assay, Labeling