Journal: Frontiers in Cardiovascular Medicine

Article Title: p90RSK-MAGI1 Module Controls Endothelial Permeability by Post-translational Modifications of MAGI1 and Hippo Pathway

doi: 10.3389/fcvm.2020.542485

Figure Lengend Snippet: MAGI1 is required to maintain EC barrier function. (A) WES analysis of MAGI1 protein expression level in lysates collected from HUVECs transduced with Ad-MAGI1 or Ad-LacZ, β-actin served as a loading control. Protein bands are shown as pseudoblots. (B) Thb (10 U/mL)-mediated reduction in TEER values observed in cells transduced with Ad-LacZ (red) was inhibited in cells transduced with Ad-MAGI1 (blue), as assessed by ECIS system and shown as normalized resistance measured approximately every 4 min for indicated times. The dashed line indicates addition of Thb. (C) Graph demonstrates normalized resistance after Thb treatment at indicated times, relative to basal level (mean ± SEM, n = 3). (D) IB analysis of MAGI1 protein expression level in lysates collected from human aortic ECs (HAECs) treated with MAGI1 siRNA (siMAGI1) (100 nM, 48 h) or control siRNA (siCTRL), tubulin served as a loading control. (E) Thb (5 U/mL)-mediated reduction in TEER values observed in cells treated with siCTRL (blue) was increased to a greater extent in cells treated with siMAGI1 (red), as assessed by the ECIS system and shown as normalized resistance measured approximately every 4 min for indicated times. The dashed line indicates addition of Thb. A reduction in TEER values indicates an increase in cell barrier permeability through paracellular mechanisms . (F) Graph demonstrates normalized resistance after Thb treatment at indicated times, relative to basal level (mean ± SEM, n = 4). Statistical significance was assessed using ANOVA followed by Bonferroni post-hoc testing for multiple group comparison. *** P < 0.001 and * P < 0.05.

Article Snippet: MAGI1 , Santa Cruz Biotechnology , sc-100326.

Techniques: Expressing, Transduction, Control, Permeability, Comparison

Journal: Frontiers in Cardiovascular Medicine

Article Title: p90RSK-MAGI1 Module Controls Endothelial Permeability by Post-translational Modifications of MAGI1 and Hippo Pathway

doi: 10.3389/fcvm.2020.542485

Figure Lengend Snippet: MAGI1 S741 phosphorylation and de-SUMOylation regulates EC permeability. (A) Schematic illustration of MAGI1 PTMs and cellular localization. (B) IB analysis of MAGI1 protein expression level in lysates collected from HAECs transduced with Ad-LacZ, -MAGI1-WT, or -S741A phosphorylation mutant, tubulin served as a loading control. (C) Thb (2.5 U/mL)-mediated reduction in TEER values observed in cells transduced with MAGI1-WT (blue) was significantly inhibited in cells transduced with the MAGI1-S741A phosphorylation mutant (red) as assessed by the ECIS system and shown as normalized resistance measured approximately every 4 min for indicated times. The dashed line indicates addition of Thb. (D) Graph demonstrates normalized resistance after Thb treatment at indicated times, relative to basal level (mean ± SEM, n = 4) (E) HUVECs transduced with Ad-Flag-MAGI1-WT or -K931R were treated with Thb (1 h) and then immunostained with a Flag antibody (green) (F) IB analysis of MAGI1 protein expression level in lysates collected from HAECs transduced by Ad-MAGI1 K931R mutant, Ad-MAGI1-WT, and Ad-LacZ, tubulin served as a loading control. (G) Thb (2.5 U/mL)-mediated reduction in TEER values observed in cells transduced with Ad-MAGI1 WT (blue) was increased to a greater extent in cells transduced with Ad-MAGI1 K931R mutant (red), as assessed by ECIS system and shown as normalized resistance measured approximately every 4 min for indicated times. The dashed line indicates addition of Thb. A reduction in TEER indicates an increase in cell barrier permeability through paracellular mechanisms . (H) Graph demonstrates normalized resistance after Thb treatment at indicated times, relative to basal level (mean ± SEM, n = 4). Statistical significance was assessed using ANOVA followed by Bonferroni post-hoc testing for multiple group comparison. *** P < 0.001 and ** P < 0.01.

Article Snippet: MAGI1 , Santa Cruz Biotechnology , sc-100326.

Techniques: Phospho-proteomics, Permeability, Expressing, Transduction, Mutagenesis, Control, Comparison

Journal: Frontiers in Cardiovascular Medicine

Article Title: p90RSK-MAGI1 Module Controls Endothelial Permeability by Post-translational Modifications of MAGI1 and Hippo Pathway

doi: 10.3389/fcvm.2020.542485

Figure Lengend Snippet: The depletion of MAGI1 increases LATS1/2 expression, but the depletion of LATS1/2 shows no effect on MAGI1 expression. (A–C) Levels of MAGI1 and LATS1/2 mRNA expression were quantified in HAECs treated with siMAGI1 with or without siLATS1 and siLATS2, or siCTRL (mean ± SEM, n = 4). (D) Wes analysis of MAGI1 and LATS1/2 protein expression levels in cell lysates collected from HAECs treated with siMAGI1 (100 nM, 48 h), actin serves as a loading control. (E) Quantification of MAGI1 and LATS1/2 expression shown in (D) (mean ± SEM, n = 3). Statistical differences between two independent groups (E,F) were assessed using the Student t -test (two-tailed), and one-way analysis of variance followed by Bonferroni post-hoc testing for multiple group. *** P < 0.001, ** P < 0.01, and * P < 0.05.

Article Snippet: MAGI1 , Santa Cruz Biotechnology , sc-100326.

Techniques: Expressing, Control, Two Tailed Test

Journal: Frontiers in Cardiovascular Medicine

Article Title: p90RSK-MAGI1 Module Controls Endothelial Permeability by Post-translational Modifications of MAGI1 and Hippo Pathway

doi: 10.3389/fcvm.2020.542485

Figure Lengend Snippet: YAP is required in the maintenance of EC barrier function, especially in LATS1/2 depleted condition. (A) HAECs were transfected with (Left panel) either siLATS1 or siYAP, or the combination of siLATS1 and siYAP; (Right panel) either siLATS2 or siYAP, or the combination of siLATS2 and siYAP, and IB analyses of LATS1/2 and YAP protein expression levels in resulting cell lysates were performed using anti-LATS1 or 2, anti-YAP, anti-β-actin as indicated (B–E) . Thb (2.5 U/mL)-mediated reduction in TEER values observed in cells transfected with siLATS1 ( A left panel, blue in B,C ), siLATS2 ( A right panel, blue in D,E ) was increased to a greater extent by siYAP transfection, as assessed by the ECIS system and shown as normalized resistance measured approximately every 4 min for indicated times. The dashed line indicates addition of Thb. A reduction in TEER values indicates an increase in cell barrier permeability through paracellular mechanisms . (C,E) Graph demonstrates normalized resistance after Thb treatment at indicated times, relative to basal level (mean ± SEM, n = 3). Statistical significance was assessed using ANOVA followed by Bonferroni post-hoc testing for multiple group comparison. *** P < 0.001, ** P < 0.01, and * P < 0.05. (F) Scheme of MAGI1-mediated Hippo pathway regulation.

Article Snippet: MAGI1 , Santa Cruz Biotechnology , sc-100326.

Techniques: Transfection, Expressing, Permeability, Comparison

Journal: Frontiers in Cardiovascular Medicine

Article Title: p90RSK-MAGI1 Module Controls Endothelial Permeability by Post-translational Modifications of MAGI1 and Hippo Pathway

doi: 10.3389/fcvm.2020.542485

Figure Lengend Snippet: The information of antibodies used in this work.

Article Snippet: MAGI1 , Santa Cruz Biotechnology , sc-100326.

Techniques:

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Tandem mass tag (TMT) proteomic analysis of fetal lungs revealed differential expression of tight junction proteins in a rat model of congenital diaphragmatic hernia.

doi: 10.1016/j.biopha.2019.109621

Figure Lengend Snippet: Fig. 6. Western blotting analysis of tight junction protein expression in the lungs of fetal rats. (A, B) Representative immunoblotting and densitometric analysis of tight junction protein expression in the fetal lung. Results were normalized relative to the expres- sion of β-actin (n = 6 per group, *P < 0.05, vs. con- trol). Western blot analysis showed increased levels of Cldn3 and decreased levels of Magi1 and Myh9 in the CDH fetal lungs.

Article Snippet: The membranes were blocked in 5% nonfat dry milk for 60min and then incubated with rabbit polyclonal anti-CLDN3 (Proteintech, Chicago, USA), rabbit polyclonal anti-MAGI1 antibody (Absin, Shanghai, China), rabbit polyclonal anti-MYH9 (Proteintech Group, Chicago, USA), and mouse monoclonal β-actin (Proteintech Group, Chicago, USA) overnight at 4 °C.

Techniques: Western Blot, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Tandem mass tag (TMT) proteomic analysis of fetal lungs revealed differential expression of tight junction proteins in a rat model of congenital diaphragmatic hernia.

doi: 10.1016/j.biopha.2019.109621

Figure Lengend Snippet: Fig. 7. IHC analysis of tight-junction proteins in the lungs of fetal rats. (A) Representative photomicrographs of IHC-staining for Cldn3, Magi1, and Myh9 in the lung sections from CDH (Left panel) and Control (right panel) fetal rats. (Original magnification ×400, scale bar = 100 μm). Cldn3 and Magi1 were mainly expressed in the epithelial cells, and some weak staining was also found in the mesenchymal cells. Myh9 protein localized to both epithelial and mesenchymal cells. (B) Semi‑quantitative analysis of IHC staining (mean density). n = 3 per group, *P < 0.05, vs. control.

Article Snippet: The membranes were blocked in 5% nonfat dry milk for 60min and then incubated with rabbit polyclonal anti-CLDN3 (Proteintech, Chicago, USA), rabbit polyclonal anti-MAGI1 antibody (Absin, Shanghai, China), rabbit polyclonal anti-MYH9 (Proteintech Group, Chicago, USA), and mouse monoclonal β-actin (Proteintech Group, Chicago, USA) overnight at 4 °C.

Techniques: Immunohistochemistry, Control, Staining

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Tandem mass tag (TMT) proteomic analysis of fetal lungs revealed differential expression of tight junction proteins in a rat model of congenital diaphragmatic hernia.

doi: 10.1016/j.biopha.2019.109621

Figure Lengend Snippet: Fig. 8. Temporal expression of tight junction mRNA in CDH lungs. (A–C) Cldn3, Magi1, and Myh9 mRNA levels in CDH lungs were determined by quantitative RT- PCR at different time points. β-actin was used as a housekeeping gene. The results were normalized relative to the same-aged control group (n = 8 per group, *P < 0.05, vs. control at the same time point).

Article Snippet: The membranes were blocked in 5% nonfat dry milk for 60min and then incubated with rabbit polyclonal anti-CLDN3 (Proteintech, Chicago, USA), rabbit polyclonal anti-MAGI1 antibody (Absin, Shanghai, China), rabbit polyclonal anti-MYH9 (Proteintech Group, Chicago, USA), and mouse monoclonal β-actin (Proteintech Group, Chicago, USA) overnight at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: The FASEB Journal

Article Title: Magi-1 scaffolds Na V 1.8 and Slack K Na channels in dorsal root ganglion neurons regulating excitability and pain

doi: 10.1096/fj.201802454RR

Figure Lengend Snippet: PDZ binding motif regulates KNa channel expression. A) Amino acid alignment of the distal C termini from orthologous Slack subunits (Xenopus, chicken, rat, and human Slack) and the rat Slick subunit. The final 4 evolutionarily conserved amino acids (ETQL) (red) represent a consensus type 1 PDZ motif (X–S/T–X–V/L/I). Green, AP-2 binding site; magenta, putative PKA phosphorylation site; blue, putative PKC phosphorylation site. B) Representative current traces of Slack and mutated Slack channels (Mut) where the PDZ motif was truncated and recombinantly expressed in CHO cells with or without Magi-1 (top). Current density analysis for each experimental condition (bottom). For each experimental condition, currents from 20 to 25 cells were analyzed. Recordings were performed 48 h after transfection. Values are expressed as ± sem. *P < 0.05 vs. respective controls. C) Representative immunoblots from Co-IP between Magi-1 and Slack when recombinantly expressed in CHO cells. D) Co-IP assay of Magi-1 with WT and a mutant Slack variant with a truncated PDZ motif. Truncating the Slack PDZ motif prevented Co-IP with Magi-1. E) Representative immunoblot of surface biotinylation assay from CHO cells coexpressing Magi-1 with Slack or Slack alone (left). Quantification of surface Slack expression is shown on the right. Data was normalized to input to account for transfection efficiency. t6 = 4.276, n = 4 per group, 2-tailed t test. *P < 0.0129. F) Double immunolabeling experiments showing overlapping expression between Magi-1 (green) (Flag antibody) and Slack (red) (top) and Magi-1 (green) (pAb) and Slack (red) (bottom) when coexpressed in CHO cells. Original magnification value, ×20. G) Representative immunoblots of Co-IP assay between Magi-1 and Slack from intact DRG neurons from adult mice. H) Double immunolabeling experiments depicting colocalization between Magi-1 (green) (pAb) and Slack (red) in cultured DRG neurons. Scale bars, 50 μm. IP, Co-Immunoprecipitation; WB, Western Blot.

Article Snippet: DRG neuron small interfering RNA transfection Small interfering RNA (siRNA) directed against Magi-1 was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Binding Assay, Expressing, Transfection, Western Blot, Co-Immunoprecipitation Assay, Mutagenesis, Variant Assay, Surface Biotinylation Assay, Immunolabeling, Cell Culture, Immunoprecipitation

Journal: The FASEB Journal

Article Title: Magi-1 scaffolds Na V 1.8 and Slack K Na channels in dorsal root ganglion neurons regulating excitability and pain

doi: 10.1096/fj.201802454RR

Figure Lengend Snippet: Magi-1 regulates Slick channels in CHO cells. A) Representative current traces of Slick currents recombinantly expressed with or without Magi-1 in CHO cells (top). Current density analysis of Slick currents for each condition (bottom). A total of 25 cells were analyzed, and values are expressed as ± sem. *P < 0.05 vs. respective controls. B) Immunoblot depicting total increased Slick protein expression during coexpression with Magi-1. Results were taken from 3 independent cultures, and values are expressed as means ± sem (t4 = 6.152, n = 3 cultures per group, 2-tailed t test). **P < 0.0021. C) Immunolabeling of recombinant Slick channels (red) and Magi-1 (green) when expressed alone or in combination in CHO cells. Scale bars, 50 μm.

Article Snippet: DRG neuron small interfering RNA transfection Small interfering RNA (siRNA) directed against Magi-1 was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Western Blot, Expressing, Immunolabeling, Recombinant

Journal: The FASEB Journal

Article Title: Magi-1 scaffolds Na V 1.8 and Slack K Na channels in dorsal root ganglion neurons regulating excitability and pain

doi: 10.1096/fj.201802454RR

Figure Lengend Snippet: Magi-1 knockdown decreases ionic currents and excitability in DRG neurons. A) Representative Magi-1 immunolabeling from cultured DRG neurons 3 d after transfection with Magi-1–targeting siRNA and nontargeting scrambled siRNA (left) using a previously validated polyclonal Magi-1 antibody. Quantification of Magi-1 immunoreactivity is shown on the right. The integrated fluorescence intensity was calculated as the product of the area and the mean pixel intensity using Metamorph software. Values from 4 independent DRG neuronal cultures per experimental condition were analyzed. Values are expressed as means ± sem [ANOVA, F(2,11) = 32.25]. Scale bar, 50 μm. ***P < 0.001 vs. respective controls. B) Representative immunoblots depicting Magi-1 expression after siRNA-mediated Magi-1 knockdown. Magi-1 antibodies normally detect multiple splice variants as indicated by the multiple bands observed on Western blot. Quantification of Magi-1 knockdown in DRG neurons (right). Three different cultures per experimental condition were analyzed. Values expressed as means ± sem [ANOVA, F(2,6) = 42.94]. ***P < 0.001 vs. respective controls. C) Representative immunoblots of surface biotinylation from DRG neurons after Magi-1 knockdown (left). Quantification of Slack channel surface expression is shown on the right. Three independent cultures were analyzed, and values are expressed as means ± sem [ANOVA, F(2,6) = 10.84]. **P < 0.01 vs. respective controls. D) Representative current traces of IK in DRG neurons after Magi-1 knockdown (top). A total of 11–12 neurons per experimental condition were analyzed, and values are expressed as means ± sem. *P ≤ 0.05. E) Representative Action Potential (AP) firing from neurons after siRNA-mediated Magi-1 knockdown during suprathreshold current stimulation (400 pA) for 1000 ms, untransduced (10 out of 10), scrambled DRG neurons 12 out of 12 fire 1 AP, whereas 12 out of 18 neurons transfected with Magi-1 siRNA failed to fire a single AP. A.u., arbitrary unit.

Article Snippet: DRG neuron small interfering RNA transfection Small interfering RNA (siRNA) directed against Magi-1 was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Immunolabeling, Cell Culture, Transfection, Fluorescence, Software, Western Blot, Expressing

Journal: The FASEB Journal

Article Title: Magi-1 scaffolds Na V 1.8 and Slack K Na channels in dorsal root ganglion neurons regulating excitability and pain

doi: 10.1096/fj.201802454RR

Figure Lengend Snippet: Magi-1 knockdown decreases NaV1.8 plasma membrane expression. A) Representative whole-cell voltage clamp current traces of total INa and TTX-resistant INa in cultured DRG neurons 3 d after transfection with Magi-1–targeting siRNA or nontargeting scrambled siRNA. B) Current density analysis of INa with different conditions. Sodium currents in neurons were recorded in either the presence or absence of 250 nM TTX. The total and TTX-resistant INa was significantly reduced after siRNA-mediated Magi-1 knockdown in cultured DRG neurons. A total of 9–12 cells per experimental group were analyzed, and values are expressed as means ± sem. C) Quantification of peak INa and TTX-resistant peak INa (at voltage step −20 mV) after Magi-1 knockdown. A total of 9–12 cells per experimental group were analyzed, and values are expressed as means ± sem [ANOVA, F(3,26) = 66.24]. *P < 0.0106, ***P < 0.001 vs. respective controls (scrambled siRNA with or without TTX). D) Representative immunoblots from surface biotinylation experiments of DRG neurons depicting reduced NaV1.8 surface expression after Magi-1 knockdown (left). Quantification of NaV1.8 surface expression is shown on the right. For quantification, 4 independent DRG cultures per experimental condition were analyzed, and values are expressed as ± sem [ANOVA, F(2,6) = 7.319]. *P < 0.05 vs. respective controls.

Article Snippet: DRG neuron small interfering RNA transfection Small interfering RNA (siRNA) directed against Magi-1 was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, Cell Culture, Transfection, Western Blot

Journal: The FASEB Journal

Article Title: Magi-1 scaffolds Na V 1.8 and Slack K Na channels in dorsal root ganglion neurons regulating excitability and pain

doi: 10.1096/fj.201802454RR

Figure Lengend Snippet: Magi-1 is expressed in DRG neurons, the SC, the SN, and at nodes of Ranvier. A) Representative immunoblots depicting Magi-1 expression from intact DRG (left) and SC (right). Untransfected CHO cell lysates were used as the control lane. CHO cells do not endogenously express Magi-1 Supplemental Fig. S1). B) Immunolabeling images showing Magi-1 (green) expression in cultured DRG neurons (panel 1), DRG sections (panel 2 and 3), and the SC (panels 4 and 5) using a previously validated monoclonal antibody. Panels 2 and 4 depict control immunolabeling, stained with secondary antibody only. DAPI (blue) labels all nuclei of cells. Scale bars, 50 μm for DRGs and 200 μm for SCs. C) Double immunolabeling depicting Magi-1 (red) and the paranodal marker Caspr (green) in SN sections (top). Arrows indicate Magi-1 labeling at nodes of Ranvier. Insets represents high-magnification images of Magi-1 immunoreactivity at nodes (bottom). Scale bars, 20 μm (top) and 10 μm (bottom). D) Frequency distribution of Magi-1 in intact DRG neurons of varying cell body size. A total of 735 neurons from 4 mice were analyzed. Neurons larger than 800 μm2 did not show high levels of Magi-1 expression.

Article Snippet: DRG neuron small interfering RNA transfection Small interfering RNA (siRNA) directed against Magi-1 was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Western Blot, Expressing, Immunolabeling, Cell Culture, Staining, Marker, Labeling

Journal: The FASEB Journal

Article Title: Magi-1 scaffolds Na V 1.8 and Slack K Na channels in dorsal root ganglion neurons regulating excitability and pain

doi: 10.1096/fj.201802454RR

Figure Lengend Snippet: Magi-1 complexes NaV1.8 channels with Slack KNa channels in DRG neurons. A) Representative whole immunoblots from Co-IP assays demonstrating binding between Magi-1 and NaV1.8 using intact adult DRG tissue. IP product samples were run in duplicate. The polyclonal Magi-1 antibody also recognized a 50-kDa band during blotting thought to be a degradation product (as per manufacturer’s description). B) Double immunolabeling experiments demonstrate similar localization between Magi-1 (green) and NaV1.8 (red) in cultured DRG neurons (panel 1), intact DRG sections (panel 2), and the spinal cord (panel 3). Scale bars, 50 μm. C) Representative immunoblots of Co-IP between Slack and NaV1.8 from intact adult DRG neurons.

Article Snippet: DRG neuron small interfering RNA transfection Small interfering RNA (siRNA) directed against Magi-1 was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Western Blot, Co-Immunoprecipitation Assay, Binding Assay, Immunolabeling, Cell Culture

Journal: The FASEB Journal

Article Title: Magi-1 scaffolds Na V 1.8 and Slack K Na channels in dorsal root ganglion neurons regulating excitability and pain

doi: 10.1096/fj.201802454RR

Figure Lengend Snippet: In vivo Magi-1 knockdown attenuates thermal nociception and acute inflammatory pain behavior. A) Experimental timeline before and after Magi-1 knockdown in vivo. B) Hargreaves test for thermal nociception showed increased PWL in ipsilateral paw injected with Magi-1–targeting shRNA when compared with the contralateral paw. No significant difference was seen in PWL between paws in mice injected with nontargeting shRNA. Behavior was taken from 9 different animals (3 females and 6 males) per experimental condition and analyzed (9). Values are expressed as means ± sem. ****P < 0.001 vs. respective controls. C) Difference score analysis determined a ∼3-s difference in withdrawal latency between ipsilateral and contralateral paw after Magi-1 shRNA in vivo transfection (d 7, 11, and 15). Values are expressed as means ± sem. *P < 0.05 vs. control. D) Formalin-induced Phase II inflammatory pain, as measured by 3 nocifensive behaviors [paw licking (left), lifting (middle), and whole-body flinches (right)] in each interval of 5 min, is reduced in mice injected with Magi-1–targeting shRNA after 15 d as compared with controls. Behavior from 9 different animals (n = 9) per experimental condition was analyzed, and values are expressed as means ± sem [ANOVA, licking: F(1,16) = 7.545; lifting: F(1,16) = 11.67; flinching: F(1,16) = 5.007]. *P < 0.05, **P < 0.01, ***P < 0.001 vs. respective controls. E) Representative Magi-1 immunolabeling in DRG sections obtained from 1 mouse injected with Magi-1–targeting shRNA (bottom left) compared with 1 mouse injected with nontargeting scrambled shRNA (top left). Magi-1 immunoreactivity was significantly reduced in ipsilateral paw from mice injected with Magi-1 shRNA as compared with contralateral paw (right). No significant change in immunoreactivity was observed in mice injected with nontargeting scrambled shRNA. DRGs from 3 different animals were analyzed, and values are expressed as means ± sem [ANOVA, F(3,20) = 9.872]. Scale bars, 50 µm. **P < 0.01 vs. respective controls. F) Western blot analysis confirmed Magi-1 knockdown in DRGs 15 d after in vivo transfection of Magi-1–targeting shRNA (left). Quantification of Western blot is shown on the right. Intact DRGs from 3 different animals were analyzed, and values are expressed as means ± sem. [ANOVA, F(3,8) = 5.161]. *P < 0.05 vs. respective controls. A.u., arbitrary unit; contra, contralateral; ipsi, ipsilateral.

Article Snippet: DRG neuron small interfering RNA transfection Small interfering RNA (siRNA) directed against Magi-1 was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: In Vivo, Injection, shRNA, Transfection, Immunolabeling, Western Blot

Journal: The FASEB Journal

Article Title: Magi-1 scaffolds Na V 1.8 and Slack K Na channels in dorsal root ganglion neurons regulating excitability and pain

doi: 10.1096/fj.201802454RR

Figure Lengend Snippet: NaV1.8 expression decreases after Magi-1 knockdown in vivo. A) Representative immunolabeling of SN depicting NaV1.8 (red) expression in paw injected with nontargeting shRNA after 15 d (top); expression of NaV1.8 at nodes of Ranvier was detected using the paranodal marker Caspr (green). Boxed areas shown are a high-magnification image of NaV1.8 and Caspr immunoreactivity (original magnification value, ×63). NaV1.8 immunoreactivity was absent in SN and at nodes in paw injected with Magi-1–targeting shRNA after 15 d (bottom). Scale bars, 20 µm. B) Representative immunoblots of NaV1.8 expression from ipsilateral and contralateral DRG lysates of mice injected in the SN with nontargeting Magi-1 shRNA (scrambled) or Magi-1–targeting shRNA. Representative blot shown for each condition is taken from the same mice. C) Quantification of NaV1.8 expression is shown on the right. Lumbar DRGs from 3 different animals were analyzed, and values are expressed as ± sem. *P < 0.05 vs. representative controls. Contra, contralateral; ipsi, ipsilateral.

Article Snippet: DRG neuron small interfering RNA transfection Small interfering RNA (siRNA) directed against Magi-1 was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, In Vivo, Immunolabeling, Injection, shRNA, Marker, Western Blot

Journal: The FASEB Journal

Article Title: Magi-1 scaffolds Na V 1.8 and Slack K Na channels in dorsal root ganglion neurons regulating excitability and pain

doi: 10.1096/fj.201802454RR

Figure Lengend Snippet: Cell-penetrating WW motif peptidomimetics alter neuronal excitability and affect pain behavior. A) Representative voltage clamp recordings depicting decreased INa (arrow) in cultured DRG neurons after 24 h of pretreatment with the peptide mimetic designated PY peptide, whereas the phospho-PY peptide increasd INa (top). Representative AP traces from cultured DRG neurons pretreated with PY peptide or phospho-PY peptide for 24 h during suprathreshold stimulation (400 pA) for 1000 ms (bottom). B) Peak INa (at voltage step −20 mV) with different peptide treatments in DRG neurons. Neurons were treated for 6 or 24 h with PY peptide or phospho-PY peptide. A total of 10–12 DRG neurons per experimental condition were analyzed, and values are expressed as means ± sem [ANOVA, F(4,35) = 19.11]. *P < 0.05, ***P < 0.001 vs. respective controls. C) NaV1.8 protein expression was altered after peptidomimetic treatment. Representative Western blot of total and surface NaV1.8 membrane expression after DRG neurons were treated with PY peptide, phospho-PY peptide, or a scrambled peptide (left) for 24 h. Quantification of Western blots shown to the right. Treatment with the PY peptide produced a significant reduction of both total and surface NaV1.8 expression when compared with scrambled peptide. The phospho-PY peptide increased surface expression of NaV1.8 when compared with scrambled peptide. Data from 3 independent cultures were analyzed, and values are expressed as means ± sem. *P < 0.05, **P < 0.01 vs. control. #P < 0.01 vs. phospho-PY peptide. D) Phase II formalin inflammatory pain was measured by nocifensive behaviors [paw licking (left), lifting (middle), and whole-body flinches (right)] in each interval of 5 min, is reduced by intraplantar pretreatment (24 h) with 100 μM (20 μl) of PY peptide, whereas phospho-PY peptide increased nocifensive behavioral responses compared with scrambled peptide control. Peptides were administered 24 h before the formalin injection (5%, 25 μl). Behavior from 6 different animals per experimental condition was analyzed, and values are expressed as means ± sem. *P < 0.05, **P < 0.01 vs. controls. #P < 0.05, ##P < 0.01 vs. phospho-PY peptide. E) Magi-1 constitutes the sodium signalosome in DRG neurons. Slack KNa channels were previously shown to internalize by AP2-CME. AP-2, adaptor complex; CL, clathrin.

Article Snippet: DRG neuron small interfering RNA transfection Small interfering RNA (siRNA) directed against Magi-1 was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Cell Culture, Expressing, Western Blot, Produced, Injection

Journal:

Article Title: Structural basis for controlling the dimerization and stability of the WW domains of an atypical subfamily

doi: 10.1110/ps.035329.108

Figure Lengend Snippet: (A) The neighbor-joining (NJ) tree dendrogram of the WW domains from human origin. The WW domain sequences from human origin were obtained from the Pfam server (http://www.sanger.ac.uk/Software/Pfam/), and the redundancy was removed using the 100% sequence identity filter. The sequences thus selected were analyzed using the program CLUSTAL W (Thompson et al. 1994). (Magenta) The members of the subclass that contains SAV1 WW2 and MAGI1 WW2. (B) Comparative representation of the LOGO plot of the representative WW domain sequences and the sequences of the members of the subclass that contains SAV1 WW2 and MAG1 WW2. (Yellow boxes) Polar residues of E242 and S246 in SAV1 WW2, as well as those of their counterparts in MAGI1 WW2, E366 and D370. (Red) Acidic residues, (blue) basic residues.

Article Snippet: The DNA encoding the human MAGI1 gene was subcloned by PCR from the human cDNA clone locus NM_004742 (TrueClone Collection, OriGene, Cat No. SC117150).

Techniques: Software, Sequencing

Journal:

Article Title: Structural basis for controlling the dimerization and stability of the WW domains of an atypical subfamily

doi: 10.1110/ps.035329.108

Figure Lengend Snippet: Equilibrium analytical ultracentrifugation of MAGI1 WW2 355–390. (Bottom panel) Radial distribution of the absorbance in the centrifuge cell at equilibrium at 16,000 rpm, with a protein concentration of 0.5 mg/mL. The solid line through the data represents the fit to a single species with a molecular weight corresponding to 5611, while the theoretical molecular weight for the single polypeptide chain is 5580. (Upper panels) Residuals for the fit.

Article Snippet: The DNA encoding the human MAGI1 gene was subcloned by PCR from the human cDNA clone locus NM_004742 (TrueClone Collection, OriGene, Cat No. SC117150).

Techniques: Analytical Ultracentrifugation, Protein Concentration, Molecular Weight

Journal:

Article Title: Structural basis for controlling the dimerization and stability of the WW domains of an atypical subfamily

doi: 10.1110/ps.035329.108

Figure Lengend Snippet: (A) The 1H-15N HSQC spectrum of MAGI1 WW2 355–390, with labels. (*) Signals from the N- and C-terminal artificial tags of the present construct. (B) Solution structures of MAGI1 WW2 355–390, in line representations. (Cyan) Residues in the N- and C-terminal artificial tags of the present construct.

Article Snippet: The DNA encoding the human MAGI1 gene was subcloned by PCR from the human cDNA clone locus NM_004742 (TrueClone Collection, OriGene, Cat No. SC117150).

Techniques: Construct

Journal:

Article Title: Structural basis for controlling the dimerization and stability of the WW domains of an atypical subfamily

doi: 10.1110/ps.035329.108

Figure Lengend Snippet: Structural comparison between mSAV1 WW2 231–266 and hMAGI1 WW2 355–390. (A) Structure of SAV1 WW2 231–266. (Lines) Side chains of key aromatic residues (F249, Y252, H256, Y263) buried in the interface of the homodimer; (neon bars) side chains of the polar residues (E242, S246). These side chains are colored to clarify the representation of the molecular interaction. (B) Structure of MAGI1 WW2 355–390. (Lines) Side chains of the aromatic residues (Y373, Y376, H380, Y387); (neon bars) side chains of the polar residues (E366, D370).

Article Snippet: The DNA encoding the human MAGI1 gene was subcloned by PCR from the human cDNA clone locus NM_004742 (TrueClone Collection, OriGene, Cat No. SC117150).

Techniques: Comparison

Journal:

Article Title: Structural basis for controlling the dimerization and stability of the WW domains of an atypical subfamily

doi: 10.1110/ps.035329.108

Figure Lengend Snippet: (A) The 1H-15N HSQC spectrum of MAGI1 WW2 355–401, with labels. (*) Signals from the N- and C-terminal artificial tags of the present construct. (B) Solution structures of MAGI1 WW2 355–401, in line representations. (Cyan) Residues in the N- and C-terminal artificial tags of the present construct.

Article Snippet: The DNA encoding the human MAGI1 gene was subcloned by PCR from the human cDNA clone locus NM_004742 (TrueClone Collection, OriGene, Cat No. SC117150).

Techniques: Construct

Journal:

Article Title: Structural basis for controlling the dimerization and stability of the WW domains of an atypical subfamily

doi: 10.1110/ps.035329.108

Figure Lengend Snippet: The 15N spin relaxation profiles of MAGI1 WW2 355–390 (black), MAGI1 WW2 355–401 (red), and MAGI1 WW2 355–390 D370S (green). The heteronuclear NOE (top), longitudinal (R1; middle), and transverse (R2; bottom) relaxation profiles are shown with error bars. (Cyan arrows) β-strands, (red bar) the α-helix of the longer construct, MAGI1 WW2 355–401.

Article Snippet: The DNA encoding the human MAGI1 gene was subcloned by PCR from the human cDNA clone locus NM_004742 (TrueClone Collection, OriGene, Cat No. SC117150).

Techniques: Construct

Journal:

Article Title: Structural basis for controlling the dimerization and stability of the WW domains of an atypical subfamily

doi: 10.1110/ps.035329.108

Figure Lengend Snippet: Thermal stability summary of MAGI1 WW2 355–390 and its variants

Article Snippet: The DNA encoding the human MAGI1 gene was subcloned by PCR from the human cDNA clone locus NM_004742 (TrueClone Collection, OriGene, Cat No. SC117150).

Techniques:

Journal:

Article Title: Structural basis for controlling the dimerization and stability of the WW domains of an atypical subfamily

doi: 10.1110/ps.035329.108

Figure Lengend Snippet: (A) Far-UV CD spectra of MAGI1 WW2 355–390 (black), MAGI1 WW2 355–401 (red), and MAGI1 WW2 355–390 D370S (green), recorded at 20°C. (B) Thermal denaturation profiles of MAGI1 WW2 355–390 (black), MAGI1 WW2 355–401 (red), and those of the D370S mutant, recorded with 55 μM (green) and 5 μM (blue) protein concentrations. The changes in ellipticity at 232 nm for MAGI1 WW2 355–390 and MAGI1 WW2 355–390 D370S, and that at 217 nm for MAGI1 WW2 355–401, were monitored and analyzed as a function of temperature.

Article Snippet: The DNA encoding the human MAGI1 gene was subcloned by PCR from the human cDNA clone locus NM_004742 (TrueClone Collection, OriGene, Cat No. SC117150).

Techniques: Circular Dichroism, Mutagenesis

Journal:

Article Title: Structural basis for controlling the dimerization and stability of the WW domains of an atypical subfamily

doi: 10.1110/ps.035329.108

Figure Lengend Snippet: (A) Close-up view highlighting the interactions around the C-terminal α-helix in MAGI1 WW2 355–401. Key residues described in the main text are marked. (B) Structural comparison between MAGI1 WW2 355–390 (blue) and MAGI1 WW2 355–401 (red). Structures were superimposed using the backbone heavy atoms in the region between Leu361 and Pro390. (C) Comparison of the accessible surface areas of the residues between MAGI1 WW2 355–390 (black) and MAGI1 WW2 355–401 (red). (Cyan bars) β-strands; (red bar) the α-helix of the longer construct, MAGI1 WW2 355–401.

Article Snippet: The DNA encoding the human MAGI1 gene was subcloned by PCR from the human cDNA clone locus NM_004742 (TrueClone Collection, OriGene, Cat No. SC117150).

Techniques: Comparison, Construct