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

Journal: The Journal of Biological Chemistry

Article Title: Identification of MKRN1 as a second E3 ligase for Eag1 potassium channels reveals regulation via differential degradation

doi: 10.1016/j.jbc.2021.100484

Figure Lengend Snippet: The MKRN1 long isoform binds to Eag1. A , co-immunoprecipitation of Eag1 and MKRN1 in HEK293T cells. Lysates from HEK293T cells coexpressing rat Eag1 with the Myc vector, Myc-tagged MKRN1 long isoform (Myc-MKRN1), or Myc-tagged MKRN1 short isoform (Myc-MKRN1-S) were immunoprecipitated (IP) with the anti-Myc (α-Myc) ( left ) or anti-Eag1 (α-Eag1) ( right ) antibodies, followed by immunoblotting with the α-Myc, α-Eag1, or anti-GAPDH (α-GAPDH) antibodies. Corresponding expression levels of Eag1 and Myc-MKRN1 in the lysates are shown in the Input lane. Henceforward, input volumes correspond to 5% of the total cell lysates used for immunoprecipitation. The positions of molecular mass markers (in kilodalton, kDa) are indicated to the left. The protein bands corresponding to Myc-MKRN1 or Myc-MKRN1-S are highlighted with arrow and arrowhead , respectively. Asterisk indicates IgG heavy chain. The expression of GAPDH was used as the loading control. B , GST pull-down assay with rat brain lysates. Top , schematic representation of the GST-MKRN1-C1 fusion protein (encoding MKRN1 amino acids 355–481). Bottom , representative immunoblot. Brain lysates were subject to the pull-down assay with the GST protein or the GST-MKRN1-C fusion protein, followed by immunoblotting with the anti-GST (α-GST) or α-Eag1 antibodies. C , representative confocal micrographs for in situ proximity ligation assay (PLA) in HEK293T cells coexpressing Myc-Eag1 with the Flag vector, Flag-MKRN1, or Flag-MKRN1-S. Fixed cells were stained with both α-Myc and α-Flag under the permeabilized configuration. In all conditions, a pair of oligonucleotide-linked Duolink PLA secondary antibodies was applied. The fluorescence signal ( red ) was only detected when the oligonucleotide labeled α-Myc and α-Flag were in close proximity. Cells were also stained with the nuclear counterstain DAPI ( blue ; right panels ). Cells were imaged in both the fluorescence ( PLA ; left panels ) and the differential interference contrast ( DIC ; center panels ) modes. D , GST pull-down assay with HEK293T lysates. Top , structural topology of the Eag1 channel and the GST-Eag1 fusion proteins. In Eag1, the cytoplasmic N-terminal region contains a Per-Arnt-Sim (PAS) domain, and the cytoplasmic C-terminal region includes three structural domains: the carboxyl linker (C-linker), the cyclic nucleotide-binding homology domain (CNBHD), and the post-CNBHD region. The amino acid range of various GST-Eag1 fusion proteins: GST-N207 (1–207), GST-C0 (493–962), GST-C1 (493–724), GST-C2 (723–848), GST-C3 (835–962), GST-C1A (493–560), GST-C1B (561–722). Bottom , representative immunoblots. Cell lysates prepared from HEK293T cells expressing Myc-MKRN1 were used for GST pull-down assay with GST or the indicated GST-Eag1 fusion proteins, followed by immunoblotting with α-Myc or α-GST. Arrows denote Myc-MKRN1 protein bands.

Article Snippet: Cells were then immunolabeled with the following primary antibodies at 4 °C for 16 h: rabbit anti-Eag1 antibody (Alomone; 1:1000).

Techniques: Immunoprecipitation, Plasmid Preparation, Western Blot, Expressing, Pull Down Assay, In Situ, Proximity Ligation Assay, Staining, Fluorescence, Labeling, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Identification of MKRN1 as a second E3 ligase for Eag1 potassium channels reveals regulation via differential degradation

doi: 10.1016/j.jbc.2021.100484

Figure Lengend Snippet: MKRN1 interacts with immature Eag1. A , MKRN1 induces the presence of Eag1 protein band c. Eag1 was coexpressed with the Myc vector, Myc-MKRN1, or Myc-MKRN1-S in HEK293T cells. Eag1 expression manifests as three distinct protein bands ( a , b , c ) of 110 to 120 kDa. B , protein band c corresponds to nonglycosylated Eag1. Cell lysates prepared from HEK293T cells expressing Eag1 or the Eag1-glycosylation mutant (Eag1-QQ) were employed for deglycosylation assays with Endo H or PNGase F: “band a” is PNGase F-sensitive but Endo H-insensitive; “band b” is PNGase F- and Endo H-sensitive; “band c” shares the same apparent molecular weight with the Eag1-QQ mutant. C , MKRN1 displays higher binding affinity to immature Eag1. Left , representative co-immunoprecipitation (CoIP) data. Eag1 was coexpressed with the Myc vector or Myc-MKRN1 in HEK293T cells. Cell lysates were immunoprecipitated with α-Myc. Right , quantification of relative CoIP efficiency. Protein densities of IP mature and immature Eag1 signals were standardized by those of corresponding Input signals, followed by normalization with respect of immature Eag1 (band b+c) (∗ p < 0.05; n = 3). D , MKRN1 binds to nonglycosylated Eag1. Left , representative CoIP data. Myc-tagged Eag1 or Eag1-QQ was coexpressed with Flag-tagged MKRN1 in HEK293T cells. Cell lysates were immunoprecipitated with α-Flag. Right , quantification of relative CoIP efficiency. Data were normalized with respect to immature Eag1 (band b+c) (n = 3).

Article Snippet: Cells were then immunolabeled with the following primary antibodies at 4 °C for 16 h: rabbit anti-Eag1 antibody (Alomone; 1:1000).

Techniques: Plasmid Preparation, Expressing, Mutagenesis, Molecular Weight, Binding Assay, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Identification of MKRN1 as a second E3 ligase for Eag1 potassium channels reveals regulation via differential degradation

doi: 10.1016/j.jbc.2021.100484

Figure Lengend Snippet: MKRN1 downregulates Eag1 protein level. A , siRNA knockdown of endogenous MKRN1 in HEK293T cells. HEK293T cells overexpressing Eag1 were subject to treatment with a negative control siRNA (si-Neg), or a siRNA specific for MKRN1 (si-MKRN1). Quantitative analyses 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 si-Neg control (∗ p < 0.05; n = 4). B , shRNA knockdown of endogenous MKRN1 in HEK293T cells. HEK293T cells overexpressing Eag1 were subject to treatment with a control shRNA for GFP (sh-GFP), or two types of shRNA specific for MKRN1 (sh-MKRN1#1, sh-MKRN1#2). Quantitative analyses are based on normalization with respect to the sh-GFP control (∗ p < 0.05; n = 4). C , shRNA knockdown of endogenous MKRN1 in cortical neurons. Cultured rat neurons were subject to treatment with a control shRNA for GFP (sh-GFP), or two types of shRNA specific for MKRN1 (sh-MKRN1#1, sh-MKRN1#3). Quantitative analyses are based on normalization with respect to the sh-GFP control (∗ p < 0.05; n = 4). D , effect of MKRN1, MKRN1-S, or MKRN1-H307E overexpression on Eag1 protein level. Eag1 was cotransfected into HEK293T cells with the indicated constructs in the molar ratio 1:5. Quantitative analyses are based on normalization with respect to the Myc vector control (∗ p < 0.05; n = 4–5). E , effect of MKRN1, MKRN1-S, or MKRN1-H307E overexpression on Eag1 current level. Top , representative Eag1 K + current traces in the presence of the indicated constructs. The holding potential was −90 mV, and the pulse potentials ranged from −80 to +60 mV. Bottom , quantification of Eag1 whole-cell current density at +40 mV. Data were normalized with respect to the Myc vector control (∗ p < 0.05). The numbers of observations are shown in parentheses.

Article Snippet: Cells were then immunolabeled with the following primary antibodies at 4 °C for 16 h: rabbit anti-Eag1 antibody (Alomone; 1:1000).

Techniques: Negative Control, shRNA, Cell Culture, Over Expression, Construct, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Identification of MKRN1 as a second E3 ligase for Eag1 potassium channels reveals regulation via differential degradation

doi: 10.1016/j.jbc.2021.100484

Figure Lengend Snippet: MKRN1 promotes Eag1 ubiquitination. A , Left , representative immunoblot showing MKRN1-mediated Eag1 ubiquitination by HA-tagged ubiquitin (HA-Ub). Ubiquitinated Eag1 is visualized as protein smears with high molecular weights. Right , quantification of relative ubiquitinated Eag1 levels with respect to the Myc vector control (∗ p < 0.05; n = 3). B , Left , representative immunoblots comparing the effect of MKRN1, MKRN1-S or MKRN1-H307E coexpression on Eag1 ubiquitination. Right , quantification of relative ubiquitinated Eag1 levels with respect to the effect of MKRN1 co-expression (∗ p < 0.05; n = 3).

Article Snippet: Cells were then immunolabeled with the following primary antibodies at 4 °C for 16 h: rabbit anti-Eag1 antibody (Alomone; 1:1000).

Techniques: Western Blot, Plasmid Preparation, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Identification of MKRN1 as a second E3 ligase for Eag1 potassium channels reveals regulation via differential degradation

doi: 10.1016/j.jbc.2021.100484

Figure Lengend Snippet: MKRN1 mediates ER-associated proteasomal degradation of Eag1. A and B , effect of 10 μM chloroquine (CQ) or 10 μM MG132 treatment (in DMSO) on MKRN1 regulation of Eag1 protein levels in HEK293T cells. Left panels , representative immunoblots. Right panels , quantification of the effect of MKRN1 coexpression on mature (band a) or immature (band b+c) Eag1 protein levels in the absence or presence of drug treatment. Protein densities were standardized as the ratio to the cognate GAPDH signals, followed by normalization with respect to the corresponding Myc vector control (∗ p < 0.05; n = 4). C and D , subcellular fractionation patterns of Eag1 in the absence or presence of Myc-MKRN1 coexpression in HEK293T cells. Left panels , representative immunoblots. Total cell homogenates (T) were ultracentrifuged and thereafter separated into the cytosol (C) and the membrane (M) fractions. The membrane pellet fraction was further sedimented through a discontinuous sucrose gradient and subsequently divided into eight fractions, with the density gradient increasing from fraction 1 toward fraction 8. Two endogenous proteins were used as specific markers for distinct subcellular compartments in HEK293T cells: cadherin (plasma membrane; fractions 2–4) and calnexin (ER membrane; fractions 7–8). Right panels , densitometric quantification of the relative distribution (with respect to the total signal) of the indicated protein bands in each membrane-associated fraction. E , representative immunofluorescence images showing the effect of MKRN1 ( magenta ) coexpression on the subcellular localization of Eag1 ( green ) in HEK293T cells. Nuclei were counterstained with DAPI ( blue ). Cells were cotransfected with the DsRed-ER expression vector ( DsRed-ER ) ( red ) to verify ER localization. Arrowheads indicate plasma membrane staining, whereas arrows denote intracellular staining. Scale bar, 10 μm. Data are representative of at least three independent experiments.

Article Snippet: Cells were then immunolabeled with the following primary antibodies at 4 °C for 16 h: rabbit anti-Eag1 antibody (Alomone; 1:1000).

Techniques: Western Blot, Plasmid Preparation, Fractionation, Immunofluorescence, Expressing, Staining

Journal: The Journal of Biological Chemistry

Article Title: Identification of MKRN1 as a second E3 ligase for Eag1 potassium channels reveals regulation via differential degradation

doi: 10.1016/j.jbc.2021.100484

Figure Lengend Snippet: MKRN1 reduces Eag1 protein stability. A , representative immunoblots showing the protein turnover time course of Eag1 coexpressed with the Myc vector, Myc-MKRN1, or Myc-MKRN1-S in HEK293T cells. Transfected cells were subject to 100 μg/ml cycloheximide (CHX) treatment for the indicated durations. B – D , quantification of the half-life values of Eag1 protein bands in response to different coexpression conditions. Symbol colors: Myc vector, black ; MKRN1, red ; MKRN1-S, blue . Left panels , linear plot of relative Eag1 protein band signals in response to different CHX treatment durations (based on the immunoblot shown in ( A )). Protein densities were standardized as the ratio to the cognate GAPDH signals, followed by normalization with respect to the corresponding no treatment control at 0 h ( Center panels ) Linear-regression analyses ( solid lines ) of the semilogarithmic plot of the same data points shown to the left . Right panels , statistical analyses of Eag1 protein half-life values derived from multiple experiments (∗ p < 0.05; n = 4–6).

Article Snippet: Cells were then immunolabeled with the following primary antibodies at 4 °C for 16 h: rabbit anti-Eag1 antibody (Alomone; 1:1000).

Techniques: Western Blot, Plasmid Preparation, Transfection, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Identification of MKRN1 as a second E3 ligase for Eag1 potassium channels reveals regulation via differential degradation

doi: 10.1016/j.jbc.2021.100484

Figure Lengend Snippet: MKRN1 fails to affect the protein stability of mature Eag1. A , representative immunoblots showing the effect of brefeldin A (BFA) treatment on mature and immature Eag1 protein bands. HEK293T cells coexpressing Eag1 and the indicated constructs were subject to up to 12 h of BFA treatment. B , MKRN1 reverses BFA-induced enhancement of immature (band b+c) Eag1 signals. Left , linear plot of relative immature Eag1 signals in response to different BFA treatment durations (based on the immunoblot shown in ( A )). Protein densities were standardized as the ratio to the cognate GAPDH signals, followed by normalization with respect to the corresponding no treatment control at 0 h. Symbols: Myc vector, black squares ; MKRN1, red circles ; MKRN1-S, blue triangles . Right , linear-regression analyses ( solid lines ) of the semilogarithmic plot of the same data points shown to the left . C , MKRN1 does not affect the time course of BFA-induced reduction of mature (band a) Eag1 signals. Left , Linear plot of relative mature Eag1 signals in response to different BFA treatment durations (based on the immunoblot shown in ( A )). Protein densities were standardized as the ratio to the cognate GAPDH signals, followed by normalization with respect to the corresponding no treatment control at 0 h. Symbols: Myc vector, black squares ; MKRN1, red circles ; MKRN1-S, blue triangles . Center , linear-regression analyses ( solid lines ) of the semilogarithmic plot of the same data points shown to the left . Right , Statistical analyses of mature (surface) Eag1 protein half-life values derived from multiple experiments (∗ p < 0.05; n = 4).

Article Snippet: Cells were then immunolabeled with the following primary antibodies at 4 °C for 16 h: rabbit anti-Eag1 antibody (Alomone; 1:1000).

Techniques: Western Blot, Construct, Plasmid Preparation, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Identification of MKRN1 as a second E3 ligase for Eag1 potassium channels reveals regulation via differential degradation

doi: 10.1016/j.jbc.2021.100484

Figure Lengend Snippet: MKRN1 regulates protein expression of disease-associated Eag1 mutants. A , Left , representative immunoblot comparing protein expression of Eag1 wild-type (WT), G348R, I467V, and G469R mutants in HEK293T cells. Right , statistical comparisons between WT and mutants were performed for total protein level ( Eag1 total ), as well as the relative signal ratio of band a to band b ( Eag1 band a/b ) (∗ p < 0.05; n = 3). B – D , effect of CUL7 overexpression, MKRN1 overexpression, or siRNA knockdown of MKRN1 on WT and mutant Eag1 protein levels in HEK293T cells. Left panels , representative immunoblots. Right panels , quantification of relative Eag1 protein levels. Eag1 signals were standardized as the ratio to the cognate GAPDH signals, followed by normalization to the corresponding Myc-vector or siRNA control (∗ p < 0.05; n = 3).

Article Snippet: Cells were then immunolabeled with the following primary antibodies at 4 °C for 16 h: rabbit anti-Eag1 antibody (Alomone; 1:1000).

Techniques: Expressing, Western Blot, Over Expression, Mutagenesis, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Identification of MKRN1 as a second E3 ligase for Eag1 potassium channels reveals regulation via differential degradation

doi: 10.1016/j.jbc.2021.100484

Figure Lengend Snippet: A dual E3 ubiquitin ligase system mediates protein homeostasis of the Eag1 channel. In this schematic model, we propose that two E3 ubiquitin ligases, CUL7 and MKRN1, play differential roles in the maintenance of Eag1 protein homeostasis. During biogenesis of immature Eag1 at the endoplasmic reticulum (ER), newly translated nascent, nonglycosylated Eag1 goes through initial glycosylation process in the ER lumen to form a core-glycosylated intermediate (pathway 1). Misfolded nonglycosylated Eag1 is recognized by MKRN1 and eventually subject to proteasomal degradation (pathway 2). Misfolded core-glycosylated Eag1, on the other hand, is identified by both CUL7 and MKRN1, followed by degradation through the ubiquitin-proteasome system (pathway 3). In contrast, properly folded immature Eag1 may exit the ER and undergo advanced glycan structure modification at the Golgi complex (pathway 4), followed by trafficking to the plasma membrane (pathway 5). At the membrane, misfolded or aged full-glycosylated Eag1 is subject to endosome-lysosomal degradation via a CUL7-depdendent mechanism (pathway 6). Overall, while both CUL7 and MKRN1 contribute to ER quality control and prevent ER exit of misfolded immature Eag1, CUL7 is additionally in charge of peripheral quality control of mature Eag1 at the cell surface.

Article Snippet: Cells were then immunolabeled with the following primary antibodies at 4 °C for 16 h: rabbit anti-Eag1 antibody (Alomone; 1:1000).

Techniques: Modification