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: Membranes were probed overnight at 4°C with antibodies against Slack anti-mouse (1:500; NeuroMab) ( 36 ), rabbit anti–β-actin (1:500; MilliporeSigma), rabbit anti–Magi-1 (1:100; Abcam), mouse anti–Magi-1 (1:100; Novus Biologicals), mouse anti-Na V 1.8 (1:200; NeuroMab), mouse anti-Na V 1.7 (1:200; NeuroMab) ( 11 ), and mouse anti-Flag (1:500; MilliporeSigma) in 5% milk prepared in Tris-buffered saline with 0.05% Tween-20.

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: Membranes were probed overnight at 4°C with antibodies against Slack anti-mouse (1:500; NeuroMab) ( 36 ), rabbit anti–β-actin (1:500; MilliporeSigma), rabbit anti–Magi-1 (1:100; Abcam), mouse anti–Magi-1 (1:100; Novus Biologicals), mouse anti-Na V 1.8 (1:200; NeuroMab), mouse anti-Na V 1.7 (1:200; NeuroMab) ( 11 ), and mouse anti-Flag (1:500; MilliporeSigma) in 5% milk prepared in Tris-buffered saline with 0.05% Tween-20.

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: Membranes were probed overnight at 4°C with antibodies against Slack anti-mouse (1:500; NeuroMab) ( 36 ), rabbit anti–β-actin (1:500; MilliporeSigma), rabbit anti–Magi-1 (1:100; Abcam), mouse anti–Magi-1 (1:100; Novus Biologicals), mouse anti-Na V 1.8 (1:200; NeuroMab), mouse anti-Na V 1.7 (1:200; NeuroMab) ( 11 ), and mouse anti-Flag (1:500; MilliporeSigma) in 5% milk prepared in Tris-buffered saline with 0.05% Tween-20.

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: Membranes were probed overnight at 4°C with antibodies against Slack anti-mouse (1:500; NeuroMab) ( 36 ), rabbit anti–β-actin (1:500; MilliporeSigma), rabbit anti–Magi-1 (1:100; Abcam), mouse anti–Magi-1 (1:100; Novus Biologicals), mouse anti-Na V 1.8 (1:200; NeuroMab), mouse anti-Na V 1.7 (1:200; NeuroMab) ( 11 ), and mouse anti-Flag (1:500; MilliporeSigma) in 5% milk prepared in Tris-buffered saline with 0.05% Tween-20.

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: Membranes were probed overnight at 4°C with antibodies against Slack anti-mouse (1:500; NeuroMab) ( 36 ), rabbit anti–β-actin (1:500; MilliporeSigma), rabbit anti–Magi-1 (1:100; Abcam), mouse anti–Magi-1 (1:100; Novus Biologicals), mouse anti-Na V 1.8 (1:200; NeuroMab), mouse anti-Na V 1.7 (1:200; NeuroMab) ( 11 ), and mouse anti-Flag (1:500; MilliporeSigma) in 5% milk prepared in Tris-buffered saline with 0.05% Tween-20.

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: Membranes were probed overnight at 4°C with antibodies against Slack anti-mouse (1:500; NeuroMab) ( 36 ), rabbit anti–β-actin (1:500; MilliporeSigma), rabbit anti–Magi-1 (1:100; Abcam), mouse anti–Magi-1 (1:100; Novus Biologicals), mouse anti-Na V 1.8 (1:200; NeuroMab), mouse anti-Na V 1.7 (1:200; NeuroMab) ( 11 ), and mouse anti-Flag (1:500; MilliporeSigma) in 5% milk prepared in Tris-buffered saline with 0.05% Tween-20.

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: Membranes were probed overnight at 4°C with antibodies against Slack anti-mouse (1:500; NeuroMab) ( 36 ), rabbit anti–β-actin (1:500; MilliporeSigma), rabbit anti–Magi-1 (1:100; Abcam), mouse anti–Magi-1 (1:100; Novus Biologicals), mouse anti-Na V 1.8 (1:200; NeuroMab), mouse anti-Na V 1.7 (1:200; NeuroMab) ( 11 ), and mouse anti-Flag (1:500; MilliporeSigma) in 5% milk prepared in Tris-buffered saline with 0.05% Tween-20.

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: Membranes were probed overnight at 4°C with antibodies against Slack anti-mouse (1:500; NeuroMab) ( 36 ), rabbit anti–β-actin (1:500; MilliporeSigma), rabbit anti–Magi-1 (1:100; Abcam), mouse anti–Magi-1 (1:100; Novus Biologicals), mouse anti-Na V 1.8 (1:200; NeuroMab), mouse anti-Na V 1.7 (1:200; NeuroMab) ( 11 ), and mouse anti-Flag (1:500; MilliporeSigma) in 5% milk prepared in Tris-buffered saline with 0.05% Tween-20.

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: Membranes were probed overnight at 4°C with antibodies against Slack anti-mouse (1:500; NeuroMab) ( 36 ), rabbit anti–β-actin (1:500; MilliporeSigma), rabbit anti–Magi-1 (1:100; Abcam), mouse anti–Magi-1 (1:100; Novus Biologicals), mouse anti-Na V 1.8 (1:200; NeuroMab), mouse anti-Na V 1.7 (1:200; NeuroMab) ( 11 ), and mouse anti-Flag (1:500; MilliporeSigma) in 5% milk prepared in Tris-buffered saline with 0.05% Tween-20.

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

Journal: The Journal of Biological Chemistry

Article Title: Scaffold Protein Connector Enhancer of Kinase Suppressor of Ras Isoform 3 (CNK3) Coordinates Assembly of a Multiprotein Epithelial Sodium Channel (ENaC)-regulatory Complex *

doi: 10.1074/jbc.M112.389148

Figure Lengend Snippet: CNK3 positively regulates ENaC surface expression and activity. A, knockdown of endogenous CNK3 decreases ENaC activity in mpkCCDc14 cells. mpkCCDc14 cells stably expressing CNK3 or control (non-target) shRNA were plated on Transwell filters and allowed to grow to high resistance. The cell monolayers were then treated with aldosterone (1 μm for 3 h) prior to determination of ENaC activity. Shown is the -fold change in aldosterone-induced equivalent short circuit current in CNK3 shRNA-expressing cells. Also shown is a representative blot demonstrating knockdown of endogenous CNK3. ***, p < 0.001 compared with control (n = 6). B, cell surface biotinylation assay for ENaC in HEK 293T cells transiently transfected with FLAG-tagged α-, β-, and γ-mENaC subunits. After transfection, cells were treated with EZ-Link sulfo-NHS-biotin before protein extraction and recovered by affinity immobilization using NeutrAvidin-conjugated agarose beads followed by immunoblot (IB) analysis with anti-β-ENaC antibody. Shown is a graphical representation of CNK3-dependent augmentation of surface ENaC based on multiple repeats of biotinylation assays. Also shown is a representative blot demonstrating CNK3 overexpression. ***, p < 0.001 compared with control (n = 4). Error bars represent S.E. WCL, whole-cell lysate.

Article Snippet: Detection of endogenous aldosterone-induced ENaC-regulatory proteins in mpkCCD c14 cells was performed using the commercially available anti-SGK1 Ab (Sigma), anti-CNK3 Ab (Protein Tech Group Inc., Chicago, IL), anti-Nedd4-2 Ab (Abcam), and anti-Raf-1 Ab (Cell Signaling Technology).

Techniques: Expressing, Activity Assay, Knockdown, Stable Transfection, Control, shRNA, Cell Surface Biotinylation Assay, Transfection, Protein Extraction, Western Blot, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: Scaffold Protein Connector Enhancer of Kinase Suppressor of Ras Isoform 3 (CNK3) Coordinates Assembly of a Multiprotein Epithelial Sodium Channel (ENaC)-regulatory Complex *

doi: 10.1074/jbc.M112.389148

Figure Lengend Snippet: CNK3 markedly stimulates interaction of SGK1 with ENaC. A, co-immunoprecipitation assays in HEK 293T cells revealing the ability of CNK3 to markedly augment interaction of SGK1 with ENaC. Blots showing total cellular expression of the various proteins specified are also depicted. GAPDH was used as a loading control. IB, immunoblot; WCL, whole-cell lysate. Also shown is a graphical representation of the above data (B) demonstrating significantly increased SGK1-ENaC interaction in the presence of CNK3. ***, p < 0.001 compared with control (n = 4). Error bars represent S.E.

Article Snippet: Detection of endogenous aldosterone-induced ENaC-regulatory proteins in mpkCCD c14 cells was performed using the commercially available anti-SGK1 Ab (Sigma), anti-CNK3 Ab (Protein Tech Group Inc., Chicago, IL), anti-Nedd4-2 Ab (Abcam), and anti-Raf-1 Ab (Cell Signaling Technology).

Techniques: Immunoprecipitation, Expressing, Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Scaffold Protein Connector Enhancer of Kinase Suppressor of Ras Isoform 3 (CNK3) Coordinates Assembly of a Multiprotein Epithelial Sodium Channel (ENaC)-regulatory Complex *

doi: 10.1074/jbc.M112.389148

Figure Lengend Snippet: CNK3 significantly stimulates interaction of SGK1 with its target substrate, Nedd4-2. A, co-immunoprecipitation assays in transiently transfected (as specified) HEK 293T cells demonstrating that CNK3 stimulates the interaction of SGK1 with Nedd4-2. Blots showing total cellular expression of the various proteins specified are also depicted. GAPDH was used as a loading control. IB, immunoblot; WCL, whole-cell lysate. Also shown is a graphical representation of the above data (B) clearly demonstrating that CNK3 markedly augments the SGK1-Nedd4-2 interaction. ***, p < 0.001 (n = 4). Error bars represent S.E.

Article Snippet: Detection of endogenous aldosterone-induced ENaC-regulatory proteins in mpkCCD c14 cells was performed using the commercially available anti-SGK1 Ab (Sigma), anti-CNK3 Ab (Protein Tech Group Inc., Chicago, IL), anti-Nedd4-2 Ab (Abcam), and anti-Raf-1 Ab (Cell Signaling Technology).

Techniques: Immunoprecipitation, Transfection, Expressing, Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Scaffold Protein Connector Enhancer of Kinase Suppressor of Ras Isoform 3 (CNK3) Coordinates Assembly of a Multiprotein Epithelial Sodium Channel (ENaC)-regulatory Complex *

doi: 10.1074/jbc.M112.389148

Figure Lengend Snippet: CNK3 is an integral component of the ERC. A–C, co-immunoprecipitation assays in HEK 293T cells demonstrating the ability of CNK3 to physically interact with SGK1 (A), ENaC (B), and the ENaC-inhibitory proteins Raf-1 and Nedd4-2 (C). Blots depicted are representative of at least four independent experiments each, performed with the same treatment protocol. Blots showing total cellular expression of the various proteins specified are also depicted. GAPDH was used as a loading control. IB, immunoblot; WCL, whole-cell lysate.

Article Snippet: Detection of endogenous aldosterone-induced ENaC-regulatory proteins in mpkCCD c14 cells was performed using the commercially available anti-SGK1 Ab (Sigma), anti-CNK3 Ab (Protein Tech Group Inc., Chicago, IL), anti-Nedd4-2 Ab (Abcam), and anti-Raf-1 Ab (Cell Signaling Technology).

Techniques: Immunoprecipitation, Expressing, Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Scaffold Protein Connector Enhancer of Kinase Suppressor of Ras Isoform 3 (CNK3) Coordinates Assembly of a Multiprotein Epithelial Sodium Channel (ENaC)-regulatory Complex *

doi: 10.1074/jbc.M112.389148

Figure Lengend Snippet: The PDZ domain in CNK3 is essential for its ability to interact with ENaC and the key ENaC-regulatory kinase, SGK1. HEK 293T cells were transiently transfected with FLAG-tagged α-, β-, and γ-mENaC subunits and V5-tagged WT- or ΔPDZ-CNK3 with or without SGK1 as specified. Shown are co-immunoprecipitation assays demonstrating the drastically reduced ability of ΔPDZ-CNK3 to interact with ENaC (A) (n = 4) or SGK1 (B) (n = 5). Blots showing total cellular expression of the various proteins specified are also depicted. GAPDH was used as a loading control. IB, immunoblot; WCL, whole-cell lysate.

Article Snippet: Detection of endogenous aldosterone-induced ENaC-regulatory proteins in mpkCCD c14 cells was performed using the commercially available anti-SGK1 Ab (Sigma), anti-CNK3 Ab (Protein Tech Group Inc., Chicago, IL), anti-Nedd4-2 Ab (Abcam), and anti-Raf-1 Ab (Cell Signaling Technology).

Techniques: Transfection, Immunoprecipitation, Expressing, Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Scaffold Protein Connector Enhancer of Kinase Suppressor of Ras Isoform 3 (CNK3) Coordinates Assembly of a Multiprotein Epithelial Sodium Channel (ENaC)-regulatory Complex *

doi: 10.1074/jbc.M112.389148

Figure Lengend Snippet: CNK3 interacts with ENaC at the cell surface and significantly stimulates its plasma membrane abundance. A, cell surface biotinylation assays in HEK 293T cells showing a marked reduction in the ability of ΔPDZ-CNK3 to stimulate surface expression of ENaC in contrast to that of WT-CNK3. Also shown is the dependence of CNK3 on ENaC for its surface recruitment. GAPDH served as a negative control for NeutrAvidin IP (“N”) blots that primarily contained surface proteins (n = 4). Sequential cell surface biotinylation and co-immunoprecipitation assays confirm that WT-CNK3 interacts with cell surface ENaC and stimulates its plasma membrane abundance (see “Experimental Procedures” for experimental details). Blots showing total cellular expression of the various proteins specified are also depicted. In whole-cell lysates (WCL), GAPDH was used as a loading control (n = 4). B, cell surface biotinylation assays for endogenous ENaC, CNK3, and SGK1 proteins in mpkCCDc14 kidney epithelial cells. Aldosterone stimulation (1 μm for 3 h) markedly augments co-localization of ENaC and associated CNK3 and SGK1 proteins on the cell surface. Blots showing total cellular expression of the various proteins specified are also depicted. In whole-cell lysates, α-tubulin was used as a loading control (n = 4). C, graphical representation of the -fold increase in total (left bar) versus plasma membrane-associated (right bar) CNK3 in response to 3-h treatment with aldosterone. Surface expression (NeutrAvidin pulldown) and whole-cell lysate data from B were quantitated by densitometry. Bars represent signal ratios in the presence versus absence of aldosterone. Total CNK3 continued to increase at later time points, reaching a peak of ∼3-fold stimulation at 6–8 h of aldosterone treatment (not shown; see text for details). ***, p < 0.001 (n = 4). Error bars represent S.E. IB, immunoblot.

Article Snippet: Detection of endogenous aldosterone-induced ENaC-regulatory proteins in mpkCCD c14 cells was performed using the commercially available anti-SGK1 Ab (Sigma), anti-CNK3 Ab (Protein Tech Group Inc., Chicago, IL), anti-Nedd4-2 Ab (Abcam), and anti-Raf-1 Ab (Cell Signaling Technology).

Techniques: Clinical Proteomics, Membrane, Expressing, Negative Control, Immunoprecipitation, Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Scaffold Protein Connector Enhancer of Kinase Suppressor of Ras Isoform 3 (CNK3) Coordinates Assembly of a Multiprotein Epithelial Sodium Channel (ENaC)-regulatory Complex *

doi: 10.1074/jbc.M112.389148

Figure Lengend Snippet: The PDZ domain in CNK3 is essential for its ability to stimulate ENaC activity. In contrast to full-length-CNK3, ΔPDZ-CNK3 cannot restore aldosterone-induced ENaC activity in mpkCCDc14 cells stably silencing the expression of endogenous CNK3. mpkCCDc14 cells stably silencing the expression of endogenous CNK3 (shRNA4) were transiently transfected with shRNA-resistant full-length (“res-CNK3”) or ΔPDZ-CNK3 (“res-ΔPDZ-CNK3”) constructs using nucleofection. Parental mpkCCDc14 cells nucleofected with an empty vector (VC) were used as a positive control (control). Unmodified wild-type CNK3 expression construct was used as an additional control (“WT-CNK3”). Shown are a graphical representation of the Ieq measured 3 h after aldosterone stimulation (1 μm) and a representative Western blot demonstrating the rescue of the exogenously expressed shRNA-resistant WT- and ΔPDZ-CNK3-V5 constructs. *, p < 0.05; ***, p < 0.001 (n = 6). Error bars represent S.E. IB, immunoblot; WCL, whole-cell lysate.

Article Snippet: Detection of endogenous aldosterone-induced ENaC-regulatory proteins in mpkCCD c14 cells was performed using the commercially available anti-SGK1 Ab (Sigma), anti-CNK3 Ab (Protein Tech Group Inc., Chicago, IL), anti-Nedd4-2 Ab (Abcam), and anti-Raf-1 Ab (Cell Signaling Technology).

Techniques: Activity Assay, Stable Transfection, Expressing, Transfection, shRNA, Construct, Plasmid Preparation, Positive Control, Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Scaffold Protein Connector Enhancer of Kinase Suppressor of Ras Isoform 3 (CNK3) Coordinates Assembly of a Multiprotein Epithelial Sodium Channel (ENaC)-regulatory Complex *

doi: 10.1074/jbc.M112.389148

Figure Lengend Snippet: CNK3 physically interacts with α- and β-ENaC in vitro. The [35S]Met-labeled in vitro translation product for wild-type CNK3 was incubated with GST alone, GST-α-ENaC, or GST-β-ENaC as indicated. After recovery of fusion proteins on glutathione-Sepharose beads, the bound fraction was analyzed by SDS-PAGE and visualized by autoradiography. 10% of in vitro translated product used in the binding assays is represented as “Input.” Shown is a representative autoradiograph from five independent GST pulldown assays performed. Similar results were obtained in all the experiments performed.

Article Snippet: Detection of endogenous aldosterone-induced ENaC-regulatory proteins in mpkCCD c14 cells was performed using the commercially available anti-SGK1 Ab (Sigma), anti-CNK3 Ab (Protein Tech Group Inc., Chicago, IL), anti-Nedd4-2 Ab (Abcam), and anti-Raf-1 Ab (Cell Signaling Technology).

Techniques: In Vitro, Labeling, Incubation, SDS Page, Autoradiography, Binding Assay

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