Journal: Journal of Neuroscience

Article Title: KCNQ2 Is a Nodal K+ Channel

doi: 10.1523/jneurosci.4512-03.2004

Figure Lengend Snippet: Figure 1. KCNQ2 is localized to PNS nodes of Ranvier. These are images of unfixed teased fibers from adult rat sciatic nerves immunolabeled for KCNQ2 (A–D) or KCNQ3 (A, E, F) and Nav channels subunits (PanNav) (C), Kv1.2 (D, E), or E-cadherin (F). KCNQ2andKCNQ3aredistinctlylocalized(A).AsingleopticalsectionobtainedbyconfocalmicroscopyshowsthatKNCQ2(red)is localizedtotheaxonalmembrane,asshownbycomparisontoNF-Hstaining(B,green).KCNQ2iscolocalizedwithNavchannelsat nodesofRanvier(C),whereasKCNQ3(A,E,F)islocalizedatincisures(greendoublearrows)andoutermesaxons(greenarrows), where it is colocalized with E-cadherin (F). Kv1.2 is localized to the axonal membrane at juxtaparanodes (D, green double arrowheads),“juxta-mesaxons”(alignedwiththeglialinnermesaxon)(E,redarrows),andthe“juxta-incisures”(alignedwiththe inneraspectofincisures)(E,reddoublearrows),locationsthataredistinctfromthoseofKCNQ2andKCNQ3.G–I,Compoundaction potentialrecordedfrom3-month-oldratsciaticnerves.Linopirdine(100M)didnotchangetheshapeoftheCAPrecordedfrom adults(G).Bycontrast,retigabine(20M)delayedtheonsetoftheCAP(H);theseeffectswereblockedbypretreatingthenerves with linopirdine (20 M) (I). Scale bars: B, 5 m; A, C–F, 10 m.

Article Snippet: Sections and teased fibers were permeabilized by immersion in 20°C acetone for 10 min, blocked at room temperature for 1 hr with 5% fish skin gelatin (0.5% Triton X-100) in PBS, and incubated overnight at 4°C with various combinations of primary antibodies: a rabbit antisera against a peptide [KCNQ2N, diluted 1:200 (Cooper et al., 2001)] or a fusion protein [n-Q2, 1:1000 (Roche et al., 2002)] to the N-terminal region of KCNQ2, a guinea pig antiserum against the N-terminal region of KCNQ3 [KCNQ3N, 1:100 (Cooper et al., 2000, 2001)]; mouse monoclonal antibodies against NMDAR1 (1: 1000; Chemicon, Temecula, CA), Kv1.2 (1:50; Alomone Laboratories, Jerusalem, Israel), ankyrin-G [1:500 (Jenkins and Bennett, 2001; Jenkins et al., 2001)], or PanNav channels (1:50; Sigma, St. Louis, MO); rat monoclonal antibody against E-cadherin (1:50; Zymed, San Francisco, CA), or the heavy chain of neurofilament [NF-H, 1:10 (Lee et al., 1982, 1987)].

Techniques: Immunolabeling, Membrane

Journal: Journal of Neuroscience

Article Title: KCNQ2 Is a Nodal K+ Channel

doi: 10.1523/jneurosci.4512-03.2004

Figure Lengend Snippet: Figure2. KCNQ2islocalizedtoCNSnodesandinitialsegments.Theseareimagesofsections ofunfixedratspinalcord,immunolabeledforKCNQ2andNMDAR1(A),ankyrin-G(B),Nav(C), or Kv1.2 (D); the merged image in A was also stained with the nuclear counterstain 4,6- diamidino-2-phenylindole.KCNQ2labelingisconcentratedinaninitialsegment(arrowhead)of an NMDAR1-positive motoneuron found in the ventral horn of spinal cord (A). KCNQ2 is colo- calized with both ankyrin-G (B) and Nav (C) at nodes in the white matter and also at initial segments (B, C, insets are from motoneurons). By contrast, Kv1.2 is confined to the juxtapara- nodes and does not colocalize with KCNQ2 (D). Scale bars: (including insets) 10 m.

Article Snippet: Sections and teased fibers were permeabilized by immersion in 20°C acetone for 10 min, blocked at room temperature for 1 hr with 5% fish skin gelatin (0.5% Triton X-100) in PBS, and incubated overnight at 4°C with various combinations of primary antibodies: a rabbit antisera against a peptide [KCNQ2N, diluted 1:200 (Cooper et al., 2001)] or a fusion protein [n-Q2, 1:1000 (Roche et al., 2002)] to the N-terminal region of KCNQ2, a guinea pig antiserum against the N-terminal region of KCNQ3 [KCNQ3N, 1:100 (Cooper et al., 2000, 2001)]; mouse monoclonal antibodies against NMDAR1 (1: 1000; Chemicon, Temecula, CA), Kv1.2 (1:50; Alomone Laboratories, Jerusalem, Israel), ankyrin-G [1:500 (Jenkins and Bennett, 2001; Jenkins et al., 2001)], or PanNav channels (1:50; Sigma, St. Louis, MO); rat monoclonal antibody against E-cadherin (1:50; Zymed, San Francisco, CA), or the heavy chain of neurofilament [NF-H, 1:10 (Lee et al., 1982, 1987)].

Techniques: Staining

Journal: Journal of Neuroscience

Article Title: KCNQ2 Is a Nodal K+ Channel

doi: 10.1523/jneurosci.4512-03.2004

Figure Lengend Snippet: Figure3. DistributionofKCNQ3inthespinalcord.Theseareimagesofsectionsofunfixedrat spinalcord,immunostainedasindicated.A,KCNQ3stainingsurroundsthesomaofamotoneu- ronintheventralhornofthespinalcordbutdoesnotlabelthesomaitselfortheinitialsegment (arrowhead).B,LongitudinalsectionofthewhitematterstainedforKCNQ3(green)andKCNQ2 (red). Some KCNQ2-positive nodes are also KCNQ3 positive (arrowheads), whereas others are KCNQ3 negative (asterisks). Insets show transverse sections of both KCNQ3-positive (bottom) and KCNQ3-negative (top) nodes. C, Transverse section of the white matter stained for KCNQ3, ankyrin-G,andtenascin-R.KCNQ3andtenascin-Rappeartosurroundankyrin-G-positivenodes. D, Transverse section of the white matter stained for KCNQ3 and GFAP, showing partially over- lapping immunoreactivity in astrocytes (arrows). Scale bars: B, C, 5 m; A, D, 10 m.

Article Snippet: Sections and teased fibers were permeabilized by immersion in 20°C acetone for 10 min, blocked at room temperature for 1 hr with 5% fish skin gelatin (0.5% Triton X-100) in PBS, and incubated overnight at 4°C with various combinations of primary antibodies: a rabbit antisera against a peptide [KCNQ2N, diluted 1:200 (Cooper et al., 2001)] or a fusion protein [n-Q2, 1:1000 (Roche et al., 2002)] to the N-terminal region of KCNQ2, a guinea pig antiserum against the N-terminal region of KCNQ3 [KCNQ3N, 1:100 (Cooper et al., 2000, 2001)]; mouse monoclonal antibodies against NMDAR1 (1: 1000; Chemicon, Temecula, CA), Kv1.2 (1:50; Alomone Laboratories, Jerusalem, Israel), ankyrin-G [1:500 (Jenkins and Bennett, 2001; Jenkins et al., 2001)], or PanNav channels (1:50; Sigma, St. Louis, MO); rat monoclonal antibody against E-cadherin (1:50; Zymed, San Francisco, CA), or the heavy chain of neurofilament [NF-H, 1:10 (Lee et al., 1982, 1987)].

Techniques: Staining

Journal: Journal of Neuroscience

Article Title: KCNQ2 Is a Nodal K+ Channel

doi: 10.1523/jneurosci.4512-03.2004

Figure Lengend Snippet: Figure 4. Localization of KCNQ2 in the developing rat spinal cord. These are images of un- fixed longitudinal sections of spinal cord from P4 (A), P8 (B), and P15 (C) rats, as well as P21 myelin-deficient rats (D), double labeled for KCNQ2 (red) and ankyrin-G or Nav (green). At P4 (A), few nodes (arrowheads) and no initial segments (insets) are KCNQ2 positive. At P8 (B), many nodes (arrowheads) and initial segments (insets) are KCNQ2 positive. At P15 (C), nearly all nodes and initial segments are KCNQ2 positive. In the spinal cord white matter of P21 myelin-deficient rats (D), where axons are undergoing demyelination because of oligodendro- cytes cell death, KCNQ2 remained colocalized with Nav in node-like clusters. Scale bar: (includ- ing insets) 10 m.

Article Snippet: Sections and teased fibers were permeabilized by immersion in 20°C acetone for 10 min, blocked at room temperature for 1 hr with 5% fish skin gelatin (0.5% Triton X-100) in PBS, and incubated overnight at 4°C with various combinations of primary antibodies: a rabbit antisera against a peptide [KCNQ2N, diluted 1:200 (Cooper et al., 2001)] or a fusion protein [n-Q2, 1:1000 (Roche et al., 2002)] to the N-terminal region of KCNQ2, a guinea pig antiserum against the N-terminal region of KCNQ3 [KCNQ3N, 1:100 (Cooper et al., 2000, 2001)]; mouse monoclonal antibodies against NMDAR1 (1: 1000; Chemicon, Temecula, CA), Kv1.2 (1:50; Alomone Laboratories, Jerusalem, Israel), ankyrin-G [1:500 (Jenkins and Bennett, 2001; Jenkins et al., 2001)], or PanNav channels (1:50; Sigma, St. Louis, MO); rat monoclonal antibody against E-cadherin (1:50; Zymed, San Francisco, CA), or the heavy chain of neurofilament [NF-H, 1:10 (Lee et al., 1982, 1987)].

Techniques: Labeling

Journal: Journal of Neuroscience

Article Title: KCNQ2 Is a Nodal K+ Channel

doi: 10.1523/jneurosci.4512-03.2004

Figure Lengend Snippet: Figure 6. Immunoblots and immunoprecipitations. A, B, Immunoblot analysis. Membrane proteins(100g)fromratmuscle,sciaticnerve,spinalcord,andbrain,andHeLacelllysates(10 g) were separated by electrophoresis and immunoblotted for KCNQ2 (A) or KCNQ3 (B). Bands correspondingtothemolecularmassofKCNQ2andKCNQ3expressedinHeLacells(97kDa)were detected in both spinal cord and brain. KCNQ3, but not KCNQ2, was detected in sciatic nerve mem- brane.C,ImmunoprecipitationsofKCNQ2andKCNQ3.Ratopticnerveandhippocampalmembranes (200g)wereimmunoprecipitatedforKCNQ2andKCNQ3andthenimmunoblottedwithKCNQ2or KCNQ3antisera.KCNQ2andKCNQ3weredetectedinbothsamples.MWmarkersareshownontheleft (inkilodaltons).D,E,CoimmunoprecipitationsofKCNQ2andankyrin-G.Ratspinalcordmembranes (200g)wereimmunoprecipitatedforKCNQ2orankyrin-Gandthenimmunoblottedforankyrin-G (D)andKCNQ2(E).A97kDaisoformofankyrin-GwaspulleddownbytheKCNQ2.Theankyrin-G antiserumpulleddownmultipleankyrin-Gisoforms,includingthe97kDaisoform.KCNQ2(aster- isk)wasimmunoprecipitatedbyboththeankyrin-GandtheKCNQ2antisera.MWmarkersareshown ontheleft(inkilodaltons).InE,theimmunoblotforKCNQ2isshownfortwofilmexposuretimes:3min (3)and10min(10).

Article Snippet: Sections and teased fibers were permeabilized by immersion in 20°C acetone for 10 min, blocked at room temperature for 1 hr with 5% fish skin gelatin (0.5% Triton X-100) in PBS, and incubated overnight at 4°C with various combinations of primary antibodies: a rabbit antisera against a peptide [KCNQ2N, diluted 1:200 (Cooper et al., 2001)] or a fusion protein [n-Q2, 1:1000 (Roche et al., 2002)] to the N-terminal region of KCNQ2, a guinea pig antiserum against the N-terminal region of KCNQ3 [KCNQ3N, 1:100 (Cooper et al., 2000, 2001)]; mouse monoclonal antibodies against NMDAR1 (1: 1000; Chemicon, Temecula, CA), Kv1.2 (1:50; Alomone Laboratories, Jerusalem, Israel), ankyrin-G [1:500 (Jenkins and Bennett, 2001; Jenkins et al., 2001)], or PanNav channels (1:50; Sigma, St. Louis, MO); rat monoclonal antibody against E-cadherin (1:50; Zymed, San Francisco, CA), or the heavy chain of neurofilament [NF-H, 1:10 (Lee et al., 1982, 1987)].

Techniques: Western Blot, Membrane, Electrophoresis

Journal: Journal of Neuroscience

Article Title: KCNQ2 Is a Nodal K+ Channel

doi: 10.1523/jneurosci.4512-03.2004

Figure Lengend Snippet: Figure7. KCNQ2modulatestheexcitabilityofpremyelinatedfibers.A,B,Imagesofhorizon- tal sections of unfixed rat optic nerve immunolabeled for ankyrin-G and KCNQ2 (A) or KCNQ3 (B).Virtuallyallankyrin-GnodesarestronglyKCNQ2positive,butafewareweaklypositivefor KCNQ3. Scale bar, 10 m. C, E, CAPs recorded from 3-month-old rat optic nerves. Linopirdine (100 M) did not affect the CAP (n 5) (C), but retigabine (20 M) delayed the onset of the CAP(n5)(D).Theeffectsofretigabinewereblockedbypretreatingthenerveswithlinopir- dine (20 M; n 4) (E). F, G, CAPs recorded from P5, P11, P17, and 3-month-old rat optic nerves.Linopirdine(100M)increasedtheduration(F)andrefractoryperiod(G)oftheCAPat bothP5(n2)andP11(n4)butnotatP17(n2)or3months(n5).Fortherefractory period,twostimuliareapplied,andtheamplitudeofthehighestpeakofthesecondevokedCAP is measured.

Article Snippet: Sections and teased fibers were permeabilized by immersion in 20°C acetone for 10 min, blocked at room temperature for 1 hr with 5% fish skin gelatin (0.5% Triton X-100) in PBS, and incubated overnight at 4°C with various combinations of primary antibodies: a rabbit antisera against a peptide [KCNQ2N, diluted 1:200 (Cooper et al., 2001)] or a fusion protein [n-Q2, 1:1000 (Roche et al., 2002)] to the N-terminal region of KCNQ2, a guinea pig antiserum against the N-terminal region of KCNQ3 [KCNQ3N, 1:100 (Cooper et al., 2000, 2001)]; mouse monoclonal antibodies against NMDAR1 (1: 1000; Chemicon, Temecula, CA), Kv1.2 (1:50; Alomone Laboratories, Jerusalem, Israel), ankyrin-G [1:500 (Jenkins and Bennett, 2001; Jenkins et al., 2001)], or PanNav channels (1:50; Sigma, St. Louis, MO); rat monoclonal antibody against E-cadherin (1:50; Zymed, San Francisco, CA), or the heavy chain of neurofilament [NF-H, 1:10 (Lee et al., 1982, 1987)].

Techniques: Immunolabeling

Journal: BMC Medicine

Article Title: Involvement of sphingosine-1-phosphate receptor 1 in pain insensitivity in a BTBR mouse model of autism spectrum disorder

doi: 10.1186/s12916-024-03722-3

Figure Lengend Snippet: Increased expression of KCNQ2 and KCNQ5 neurons in DRG of BTBR mice. a – c Quantification of KCNQ2 , KCNQ5 , and KCNQ3 mRNA; n = 8–11 mice per group. d – f Representative western blotting results and quantification of the KCNQ2, KCNQ3, and KCNQ5 proteins. n = 8 mice per group. g and h Comparison of the percentage of KCNQ2 + and KCNQ5 + neurons in the BTBR and Con group. n = 8–14 mice per group. i and j Representative images of KCNQ2 and KCNQ5 immunofluorescence in mice DRGs. Scale bars = 100 μm. k and l Distribution of KCNQ2 + and KCNQ5 + neurons of different diameters in the Con and BTBR group. n = 5 mice per group. m XE-991 induced nociceptive behavior in BTBR mice. n = 6 mice per group. All data are shown in bar diagrams, which reflect the arithmetic mean ± standard error of the mean. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. not significantly different

Article Snippet: The monoclonal antibodies used for western blotting were as follows: anti-S1PR1 (Rabbit, 1:1000, 55,133–1-Ap, Proteintech, USA); anti-S1PR3 (Rabbit, 1:1000; NBP2-24,762, Novus, USA); anti-KCNQ2 (Guinea pig, 1:200; AGP-065, Alomone, Israel); anti-KCNQ3 (Rabbit, 1:200; APC-051, Alomone, Israel); anti-KCNQ5 (Rabbit, 1:1000; YT2460, Proteintech, USA); anti-phospho-p44/42 MAPK (Rabbit, 1:2000; 7370S, CST, USA); anti-p44/42 MAPK (Erk1/2) (Rabbit, 1:2000; 4695, CST, USA); anti-phospho-p38 MAPK (Rabbit, 1:1000; 4511S, CST, USA); anti-p38 MAPK (Rabbit, 1:1000; 8690S, CST, USA); anti-PKA (Rabbit, 1:1000; 4782S, CST, USA); anti-phospho-PKA C (Rabbit, 1:1000; 5561S, CST, USA); anti-PKC (Rabbit, 1:1000; ab181558, Abcam, UK); anti-phospho-PKC (Rabbit, 1:1000; 38938S, CST, USA); anti-GADPH (Rabbit, 1:1000; 5174S, CST, USA); anti-β-tubulin (Rabbit, 1:2000; 10,094–1-AP, Proteintech, USA); and anti-β-actin (Rabbit, 1:1000, 4970 s, CST, USA).

Techniques: Expressing, Western Blot, Comparison, Immunofluorescence

Journal: BMC Medicine

Article Title: Involvement of sphingosine-1-phosphate receptor 1 in pain insensitivity in a BTBR mouse model of autism spectrum disorder

doi: 10.1186/s12916-024-03722-3

Figure Lengend Snippet: M channel and abnormal MAPK cAMP/PKA signaling pathways rescued by inhibition of S1PR1 in mice DRGs. a – c Quantification of KCNQ2 , KCNQ3 , and KCNQ5 mRNA levels after the W146 intervention. n = 6–12 mice per group. d – f Representative western blotting results and quantification of the KCNQ2, KCNQ3, and KCNQ5 proteins after W146 intervention. n = 8 mice per group. g and h Representative images of KCNQ2 and KCNQ5 immunofluorescence in mice DRGs. Scale bars = 100 μm. Comparison of the percentage of KCNQ2 + neurons in the BTBR + W146 and BTBR group; n = 6–9 mice per group. i – l Representative western blotting results and quantification of P-ERK/ERK, P-P38/P38, P-PKA/PKA, and P-PKC/PKC proteins. n = 8 mice per group. All data are shown in bar diagrams, which reflect the arithmetic mean ± standard error of the mean. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. not significantly different

Article Snippet: The monoclonal antibodies used for western blotting were as follows: anti-S1PR1 (Rabbit, 1:1000, 55,133–1-Ap, Proteintech, USA); anti-S1PR3 (Rabbit, 1:1000; NBP2-24,762, Novus, USA); anti-KCNQ2 (Guinea pig, 1:200; AGP-065, Alomone, Israel); anti-KCNQ3 (Rabbit, 1:200; APC-051, Alomone, Israel); anti-KCNQ5 (Rabbit, 1:1000; YT2460, Proteintech, USA); anti-phospho-p44/42 MAPK (Rabbit, 1:2000; 7370S, CST, USA); anti-p44/42 MAPK (Erk1/2) (Rabbit, 1:2000; 4695, CST, USA); anti-phospho-p38 MAPK (Rabbit, 1:1000; 4511S, CST, USA); anti-p38 MAPK (Rabbit, 1:1000; 8690S, CST, USA); anti-PKA (Rabbit, 1:1000; 4782S, CST, USA); anti-phospho-PKA C (Rabbit, 1:1000; 5561S, CST, USA); anti-PKC (Rabbit, 1:1000; ab181558, Abcam, UK); anti-phospho-PKC (Rabbit, 1:1000; 38938S, CST, USA); anti-GADPH (Rabbit, 1:1000; 5174S, CST, USA); anti-β-tubulin (Rabbit, 1:2000; 10,094–1-AP, Proteintech, USA); and anti-β-actin (Rabbit, 1:1000, 4970 s, CST, USA).

Techniques: Inhibition, Western Blot, Immunofluorescence, Comparison

Journal: bioRxiv

Article Title: Impacted Spike Frequency Adaptation Associated with Reduction of KCNQ2/3 Promotes Seizure Activity in Temporal Lobe Epilepsy

doi: 10.1101/2020.09.25.313254

Figure Lengend Snippet: Two gene expression profiling databases both indicate significant decreases of KCNQ2 and KCNQ3 expressions in TLE patients’ hippocampus. a , mRNA levels of KCNQ2 in TLE hippocampus vs control, from Database 1 (see Method). b , mRNA levels of KCNQ3 in TLE hippocampus vs control from Database 1. c , mRNA levels of KCNQ2 from Database 2. d , mRNA levels of KCNQ3 from Database 2 (see Method). Significant differences are based on unpaired Students’ t-test. TLE_HS: temporal lobe epilepsy with hippocampal sclerosis.

Article Snippet: 25~30ug of total protein was resolved in 10% Nupage Bis-Tris gel and probed with followed primary antibodies: mouse monoclonal anti-KCNQ2 antibody (Proteintech, 66774-1-Ig, 1: 1,000), rabbit polyclonal anti-KCNQ3 antibody (Proteintech, 19966-1-AP, 1: 1,000) and mouse monoclonal anti-beta-actin antibody (Sigma, A5441, 1: 10,000).

Techniques: Gene Expression, Control

Journal: bioRxiv

Article Title: Impacted Spike Frequency Adaptation Associated with Reduction of KCNQ2/3 Promotes Seizure Activity in Temporal Lobe Epilepsy

doi: 10.1101/2020.09.25.313254

Figure Lengend Snippet: The relationships of mRNA levels of KCNQ2/3 and duration of epilepsy ( a ), onset age ( b ) as well as age ( c ) in Database 2 were exhibited respectively.

Article Snippet: 25~30ug of total protein was resolved in 10% Nupage Bis-Tris gel and probed with followed primary antibodies: mouse monoclonal anti-KCNQ2 antibody (Proteintech, 66774-1-Ig, 1: 1,000), rabbit polyclonal anti-KCNQ3 antibody (Proteintech, 19966-1-AP, 1: 1,000) and mouse monoclonal anti-beta-actin antibody (Sigma, A5441, 1: 10,000).

Techniques:

Journal: bioRxiv

Article Title: Impacted Spike Frequency Adaptation Associated with Reduction of KCNQ2/3 Promotes Seizure Activity in Temporal Lobe Epilepsy

doi: 10.1101/2020.09.25.313254

Figure Lengend Snippet: a , decreased protein levels of KCNQ2/3 in dentate gyrus of TLE mouse were detected by immunoblotting. b , statistical analysis of a . c , representative traces of spike firing captured from granule cells in control (blue) or TLE dentate gyrus. d and e , statistical analysis of initial frequency (f 0 , in panel d ) and steady-state frequency (f inf , in panel e ). All significant differences are based on unpaired Students’ t-test.

Article Snippet: 25~30ug of total protein was resolved in 10% Nupage Bis-Tris gel and probed with followed primary antibodies: mouse monoclonal anti-KCNQ2 antibody (Proteintech, 66774-1-Ig, 1: 1,000), rabbit polyclonal anti-KCNQ3 antibody (Proteintech, 19966-1-AP, 1: 1,000) and mouse monoclonal anti-beta-actin antibody (Sigma, A5441, 1: 10,000).

Techniques: Western Blot, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: Clinical analysis and functional characterization of KCNQ2-related developmental and epileptic encephalopathy

doi: 10.3389/fnmol.2023.1205265

Figure Lengend Snippet: Locations of the pathogenic KCNQ2 variants. (A) Topology diagram of the KCNQ2 channel. The KCNQ2 subunits possess six transmembrane segment regions (S1-S6) and a long intracellular C terminus. Segments S1–S3 and S4 form the voltage-sensing domain. S5–S6 in conjunction with their extracellular linker constitute the channel pore. C-terminal helices A and B indicate the sites for interaction with calmodulin. Helices C and D are involved in subunit–subunit interactions. The locations of the variants described in the present report are shown as circles in different colors. (B) Structure of the human KCNQ2 (PDB:7CR3) (Li et al., ). The side view is shown on the left and the top view is on the right. The variants are shown as sphere styles in different colors. The p.A185T and p.K606X are not shown because they are not resolved in the original structure.

Article Snippet: The channel subunits in total lysates and streptavidin precipitates were analyzed by Western blotting using rabbit monoclonal anti-KCNQ2 primary antibodies (D9L5S, dilution 1:1,000; Cell Signaling Technology, 14752), followed by secondary antibodies (Alexa Fluor 680 Conjugate; dilution 1:5000; Abcam, 175773).

Techniques:

Journal: Frontiers in Molecular Neuroscience

Article Title: Clinical analysis and functional characterization of KCNQ2-related developmental and epileptic encephalopathy

doi: 10.3389/fnmol.2023.1205265

Figure Lengend Snippet: Functional properties of the homomericKCNQ2 variants. (A) Macroscopic current traces recorded in CHO cells transfected with KCNQ2 variants (3μg) in homomeric configuration. Current scale, 250 pA; time scale, 200 ms. (B) Mean current densities of KCNQ2 channel at +40 mV in the homozygous state. (C) Current–voltage relationships of the KCNQ2 homomeric channel were determined from tail current amplitudes. Lines represent fits of a Boltzmann function. (D) Differences in activation of V 0.5 were determined for the KCNQ2 channel expressed in the homozygous state ( n = 8–15). Statistically significant differences * p < 0.05 versus WT KCNQ2.

Article Snippet: The channel subunits in total lysates and streptavidin precipitates were analyzed by Western blotting using rabbit monoclonal anti-KCNQ2 primary antibodies (D9L5S, dilution 1:1,000; Cell Signaling Technology, 14752), followed by secondary antibodies (Alexa Fluor 680 Conjugate; dilution 1:5000; Abcam, 175773).

Techniques: Functional Assay, Transfection, Activation Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Clinical analysis and functional characterization of KCNQ2-related developmental and epileptic encephalopathy

doi: 10.3389/fnmol.2023.1205265

Figure Lengend Snippet: Functional properties of the KCNQ2 variants coexpressed with WTKCNQ2. (A) Macroscopic current traces recorded in CHO cells transfected KCNQ2 variants with WTKCNQ2in a 1:1 ratio (1.5 μg:1.5 μg). Current scale, 250 pA; time scale, 200 ms. (B) The mean current densities of the KCNQ2 variants coexpressed with WT in a 1:1 ratio at +40mV. (C) Current–voltage relationships of KCNQ2 variants coexpressed with WTKCNQ2 in a 1:1 ratio determined from tail current amplitudes. Lines represent fits of a Boltzmann function. (D) Differences in activation of V0.5 were determined for the KCNQ2 variants and WTKCNQ2 in a 1:1 ratio ( n = 8–15). Statistically significant differences * p < 0.05 versus WT KCNQ2.

Article Snippet: The channel subunits in total lysates and streptavidin precipitates were analyzed by Western blotting using rabbit monoclonal anti-KCNQ2 primary antibodies (D9L5S, dilution 1:1,000; Cell Signaling Technology, 14752), followed by secondary antibodies (Alexa Fluor 680 Conjugate; dilution 1:5000; Abcam, 175773).

Techniques: Functional Assay, Transfection, Activation Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Clinical analysis and functional characterization of KCNQ2-related developmental and epileptic encephalopathy

doi: 10.3389/fnmol.2023.1205265

Figure Lengend Snippet: Biophysical properties of mutant KCNQ2/3 channel.

Article Snippet: The channel subunits in total lysates and streptavidin precipitates were analyzed by Western blotting using rabbit monoclonal anti-KCNQ2 primary antibodies (D9L5S, dilution 1:1,000; Cell Signaling Technology, 14752), followed by secondary antibodies (Alexa Fluor 680 Conjugate; dilution 1:5000; Abcam, 175773).

Techniques: Mutagenesis

Journal: Frontiers in Molecular Neuroscience

Article Title: Clinical analysis and functional characterization of KCNQ2-related developmental and epileptic encephalopathy

doi: 10.3389/fnmol.2023.1205265

Figure Lengend Snippet: Functional properties of the KCNQ2 variants coexpressed with WTKCNQ2 and WTKCNQ3. (A) Macroscopic current traces recorded in CHO cells transfected KCNQ2 variants with WTKCNQ2 and WTKCNQ3 in 0.5:0.5:1 ratio (0.75 μg:0.75 μg:1.5 μg). Current scale, 1 nA; time scale, 200 ms. (B) The mean current densities of the KCNQ2variants coexpressed with WTKCNQ2 and WTKCNQ3 in a 0.5:0.5:1 ratio at +40 mV. (C) Current–voltage relationships of KCNQ2 variants coexpressed with WTKCNQ2 and WTKCNQ3 in a 0.5:0.5:1 ratio determined from tail current amplitudes. Lines represent fits of a Boltzmann function. (D) Differences in activation of V 0.5 were determined for the KCNQ2 variants, WTKCNQ2 and WTKCNQ3in a 0.5:0.5:1 ratio orWTKCNQ2 and WTKCNQ3in a 0.5:1ratio ( n = 8–11). Statistically significant differences * p < 0.05 versus WTKCNQ2/WTKCNQ3 in a 1:1 ratio. # p < 0.05 vs. WTKCNQ2/WTKCNQ3 in a 0.5:1 ratio.

Article Snippet: The channel subunits in total lysates and streptavidin precipitates were analyzed by Western blotting using rabbit monoclonal anti-KCNQ2 primary antibodies (D9L5S, dilution 1:1,000; Cell Signaling Technology, 14752), followed by secondary antibodies (Alexa Fluor 680 Conjugate; dilution 1:5000; Abcam, 175773).

Techniques: Functional Assay, Transfection, Activation Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Clinical analysis and functional characterization of KCNQ2-related developmental and epileptic encephalopathy

doi: 10.3389/fnmol.2023.1205265

Figure Lengend Snippet: Total expression and surface expression of KCNQ2 channel. (A) Western blot analysis of proteins from total lysates (top) or plasma membrane fractions (bottom) from the negative control (NC, non-transfected CHO cells) or the CHO cells transfected with KCNQ2 variants. (B) Protein expression levels in total lysates obtained from transfected CHO cells were determined by normalizing KCNQ2 variants to the corresponding actin signals in four independent experiments. (C) Surface expression levels of the KCNQ2 variants normalized to WTKCNQ2 from four independent experiments. Statistically significant differences * p < 0.05 versus WT KCNQ2.

Article Snippet: The channel subunits in total lysates and streptavidin precipitates were analyzed by Western blotting using rabbit monoclonal anti-KCNQ2 primary antibodies (D9L5S, dilution 1:1,000; Cell Signaling Technology, 14752), followed by secondary antibodies (Alexa Fluor 680 Conjugate; dilution 1:5000; Abcam, 175773).

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

Journal: Cell Communication and Signaling : CCS

Article Title: Involvement of HDAC2-mediated kcnq2/kcnq3 genes transcription repression activated by EREG/EGFR-ERK-Runx1 signaling in bone cancer pain

doi: 10.1186/s12964-024-01797-2

Figure Lengend Snippet: Knockdown of HDAC2 in DRG neurons disrupts the transcriptional repression of kcnq2 and kcnq3 genes, reduces the neuronal excitability and attenuates pain hypersensitivity in bone cancer pain model rats. ( A - D ) ChIP-qPCR assays for the enrichment of acetylated histone 3 (H3Kac) and acetylated histone 4 (H4Kac) in the kcnq2 and kcnq3 genes promoter, in ipsilateral L4/5 DRG tissues obtained from bone cancer pain (BCP) model rats that received intrathecal LV-shHDAC2 or the control LV-GFP, performed at 14 days after tumor cells inoculation. ( A and B ) for H3Kac ( n = 4–6 rats per group); ( C and D ) for H4Kac ( n = 8 rats per group). ( E – H ) RT-qPCR and Western blot analyses of the mRNA and protein abundance of KCNQ2 and KCNQ3 in ipsilateral L4/5 DRG tissues obtained from BCP model rats that received intrathecal LV-shHDAC2 or the control LV-GFP, performed at 14 days after tumor cells inoculation. ( E and F ) for KCNQ2 ( n = 6–9 rats per group); ( G and H ) for KCNQ3 ( n = 6–7 rats per group). Upper in ( F and H ): Representative blots are shown. ( I - O ) Electrophysiological analyses of M-currents ( I and M ) and neuronal excitability ( J - O ) in ipsilateral L4/5 DRG neurons of BCP model rats that received intrathecal LV-shHDAC2 or the control LV-GFP, recorded at 14 days after tumor cells inoculation. ( I and J ) Representative traces of M-currents ( I ) and neuronal action potentials ( J ) evoked by a large depolarizing current pulse (1-s, 2-fold AP rheobase) are shown. Scale bar = 100 pA, 300 ms for ( I ), and 20 mV, 100 ms for ( J ). ( K and L ) Analysis of neuronal firing rate (spikes/second) elicited by a series of 500-ms depolarizing current pulses (in 50-pA steps from 0 to 400 pA). ( K ) Representative traces of evoked action potentials (APs) by 100 pA, 200 pA, and 300 pA depolarizing current pulses are shown. Scale bar = 20 mV, 200 ms. ( M – O ) Plots of M-current density, frequency of APs, inter-spike intervals (ISI), AP amplitude, after hyperpolarization (AHP) amplitude, AHP duration at 80% repolarization (AHP 80% duration) ( M ), threshold potential ( N ), and rheobase ( O ) ( n = 20–30 cells from six rats per group). ( P and Q ) Assessment of ipsilateral PWT ( P ) and PWL ( Q ) of BCP model rats that received intrathecal LV-shHDAC2 or the control LV-GFP, performed at 14 days after tumor cells inoculation ( n = 11 rats per group). ( R ) Assessment of animal’s locomotor function before and after intrathecal lentivirus administration ( n = 11 rats per group). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, unpaired t test for ( A )-( H ) and ( M )-( O ); two-way ANOVA with Sidak’s post hoc test for ( L ) and ( P )-( R ). See also Fig. S6

Article Snippet: Then, tissues or cultured cells were incubated with the corresponding primary antibody (see Supplementary Table S1) in PBS at 4 °C overnight, which includes rabbit anti-KCNQ2 (1:200, Abcam), rabbit anti-KCNQ3 (1:200, Abcam), rabbit anti-mouse NeuN (1:200, Sigma-Aldrich), mouse anti-pig GFAP (1:200, Cell Signaling Technology), mouse anti-NF200 (1:200, Sigma-Aldrich), mouse anti-CGRP (1:200, Abcam), rabbit anti-HDAC2 (1:200, Abcam), rabbit anti-MeCP2 (1:200, Abcam), goat anti-EGFR antibody (1:200, GeneTex), rabbit anti-Runx1(1:200, Abcam), goat anti-Sin3A (1:200, R&D systems).

Techniques: Knockdown, Control, Quantitative RT-PCR, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Involvement of HDAC2-mediated kcnq2/kcnq3 genes transcription repression activated by EREG/EGFR-ERK-Runx1 signaling in bone cancer pain

doi: 10.1186/s12964-024-01797-2

Figure Lengend Snippet: Knockdown of MeCP2 in DRG neurons impairs HDAC2-mediated transcriptional repression of kcnq2 and kcnq3 genes, reduces the neuronal excitability and attenuates pain hypersensitivity in bone cancer pain model rats. ( A - F ) ChIP-qPCR assays for the enrichment of MeCP2-HDAC2 complex, acetylated histone 3 (H3Kac) and acetylated histone 4 (H4Kac) in the kcnq2 and kcnq3 genes promoter, in ipsilateral L4/5 DRG tissues obtained from bone cancer pain (BCP) model rats that received intrathecal LV-shMeCP2 or the control LV-ZsGreen, performed at 14 days after tumor cells inoculation. ( A and B ) for MeCP2-HDAC2 complex ( n = 6–8 rats per group); ( C and D ) for H3Kac ( n = 6–8 rats per group); ( E and F ) for H4Kac ( n = 6–7 rats per group). ( G - J ) RT-qPCR and Western blot analyses of the mRNA and protein abundance of KCNQ2 and KCNQ3 in ipsilateral L4/5 DRG tissues obtained from BCP model rats that received intrathecal LV-shMeCP2 or LV-ZsGreen, performed at 14 days after tumor cells inoculation. ( G and H ) for KCNQ2, ( I and J ) for KCNQ3, n = 5–7 rats per group. Upper in ( H and J ): Representative blots are shown. ( K - Q ) Electrophysiological analyses of M-currents ( K and O ) and neuronal excitability ( L-Q ) in ipsilateral L4/5 DRG neurons of BCP model rats that received intrathecal LV-shMeCP2 or LV-ZsGreen, recorded at 14 days after tumor cells inoculation. ( K and L ) Representative traces of M-currents ( K ) and neuronal action potentials ( L ) evoked by a large depolarizing current pulse (1-s, 2-fold AP rheobase) are shown. Scale bar = 100 pA, 300 ms for ( K ), and 20 mV, 100 ms for ( L ). ( M and N ) Analysis of neuronal firing rate (spikes/second) elicited by a series of 500-ms depolarizing current pulses (in 50-pA steps from 0 to 400 pA). ( M ) Representative traces of evoked action potentials (APs) by 100 pA, 200 pA, and 300 pA depolarizing current pulses are shown. Scale bar = 20 mV, 200 ms. ( O - Q ) Plots of M-current density, frequency of APs, inter-spike intervals (ISI), AP amplitude, after hyperpolarization (AHP) amplitude, AHP 80% duration ( O ), threshold potential ( P ), and rheobase ( Q ) ( n = 20–30 cells from six rats per group). ( R and S ) Assessment of ipsilateral PWT ( R ) and PWL ( S ) for BCP model rats that received intrathecal LV-shMeCP2 or LV-ZsGreen, performed at 14 days after tumor cells inoculation ( n = 13–14 rats per group). ( T ) Assessment of animal’s locomotor function before and after intrathecal lentivirus administration ( n = 13–14 rats per group). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, unpaired t test for ( A )-( J ) and ( O )-( Q ); two-way ANOVA with Sidak’s post hoc test for ( N ) and ( R )-( T ). See also Fig. S7-S9

Article Snippet: Then, tissues or cultured cells were incubated with the corresponding primary antibody (see Supplementary Table S1) in PBS at 4 °C overnight, which includes rabbit anti-KCNQ2 (1:200, Abcam), rabbit anti-KCNQ3 (1:200, Abcam), rabbit anti-mouse NeuN (1:200, Sigma-Aldrich), mouse anti-pig GFAP (1:200, Cell Signaling Technology), mouse anti-NF200 (1:200, Sigma-Aldrich), mouse anti-CGRP (1:200, Abcam), rabbit anti-HDAC2 (1:200, Abcam), rabbit anti-MeCP2 (1:200, Abcam), goat anti-EGFR antibody (1:200, GeneTex), rabbit anti-Runx1(1:200, Abcam), goat anti-Sin3A (1:200, R&D systems).

Techniques: Knockdown, Control, Quantitative RT-PCR, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Involvement of HDAC2-mediated kcnq2/kcnq3 genes transcription repression activated by EREG/EGFR-ERK-Runx1 signaling in bone cancer pain

doi: 10.1186/s12964-024-01797-2

Figure Lengend Snippet: Epiregulin (EREG) mediates kcnq2 and kcnq3 genes transcriptional repression in DRG neurons, enhances neuronal excitability and induces pain hypersensitivity in naïve rats. ( A ) RT-qPCR and Western blot analyses of the mRNA and protein abundance of KCNQ2 and KCNQ3 in L4/5 DRG tissues obtained from naïve rats that received intrathecal EREG or vehicle, performed at 5 days after drug administration. ( A and B ) for KCNQ2 ( n = 5–6 rats per group); ( C and D ) for KCNQ3 ( n = 6–7 rats per group). Upper in ( B and D ): Representative blots are shown. ( E - K ) Electrophysiological analyses of M-currents ( E and I ) and neuronal excitability ( F - K ) in L4/5 DRG tissues obtained from naïve rats that received intrathecal EREG or vehicle, recorded at 5 days after drug administration. ( E and F ) Representative traces of M-currents ( E ) and neuronal action potentials ( F ) evoked by a large depolarizing current pulse (1-s, 2-fold AP rheobase) are shown. Scale bar = 100 pA, 300 ms for ( E ), and 20 mV, 100 ms for ( F ). ( G and H ) Analysis of neuronal firing rate (spikes/second) elicited by a series of 500-ms depolarizing current pulses (in 50-pA steps from 0 to 400 pA). ( G ) Representative traces of evoked action potentials (APs) by 100 pA, 200 pA, and 300 pA depolarizing current pulses are shown. Scale bar = 20 mV, 200 ms. ( I - K ) Plots of M-current density, frequency of APs, inter-spike intervals (ISI), AP amplitude, after hyperpolarization (AHP) amplitude, AHP 80% duration ( I ), threshold potential ( J ), and rheobase ( K ) ( n = 20–30 cells from six rats per group). ( L ) Assessment of the PWT for naive rats that received intrathecal EREG or vehicle, performed at 5 days after drug administration ( n = 12–13 rats per group). ( M ) Assessment of animal’s locomotor function before and after intrathecal drug administration ( n = 12–13 rats per group). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, unpaired t test for ( A )-( D ) and ( I - K ); two-way ANOVA with Sidak’s post hoc test for ( H ), ( L ), and ( M )

Article Snippet: Then, tissues or cultured cells were incubated with the corresponding primary antibody (see Supplementary Table S1) in PBS at 4 °C overnight, which includes rabbit anti-KCNQ2 (1:200, Abcam), rabbit anti-KCNQ3 (1:200, Abcam), rabbit anti-mouse NeuN (1:200, Sigma-Aldrich), mouse anti-pig GFAP (1:200, Cell Signaling Technology), mouse anti-NF200 (1:200, Sigma-Aldrich), mouse anti-CGRP (1:200, Abcam), rabbit anti-HDAC2 (1:200, Abcam), rabbit anti-MeCP2 (1:200, Abcam), goat anti-EGFR antibody (1:200, GeneTex), rabbit anti-Runx1(1:200, Abcam), goat anti-Sin3A (1:200, R&D systems).

Techniques: Quantitative RT-PCR, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Involvement of HDAC2-mediated kcnq2/kcnq3 genes transcription repression activated by EREG/EGFR-ERK-Runx1 signaling in bone cancer pain

doi: 10.1186/s12964-024-01797-2

Figure Lengend Snippet: Knockdown of EGFR in DRG neurons impairs HDAC2-mediated transcriptional repression of kcnq2 and kcnq3 genes, reduces neuronal excitability and attenuates pain hypersensitivity in bone cancer pain model rats. ( A and B ) RT-qPCR and Western blot analyses of HDAC2 mRNA ( A ) and protein ( B ) abundance in ipsilateral L4/5 DRG tissues obtained from bone cancer pain (BCP) model rats that received intrathecal LV-shEGFR or the control LV-ZsGreen, performed at 14 days after tumor cells inoculation ( n = 6–8 rats per group). Upper in ( B ): Representative blots are shown. ( C - H ) ChIP-qPCR assays for the enrichment of HDAC2, acetylated histone 3 (H3Kac), and acetylated histone 4 (H4Kac) in the kcnq2 and kcnq3 genes promoter, in ipsilateral L4/5 DRG tissues obtained from bone cancer pain (BCP) model rats that received intrathecal LV-shEGFR or the control LV-ZsGreen, performed at 14 days after tumor cells inoculation. ( C and D ) for HDAC2 ( n = 5–6 rats per group); ( E and F ) for H3Kac ( n = 5 rats per group); ( G and H ) for H4Kac ( n = 5–6 rats per group). ( I - L ) RT-qPCR and Western blot analyses of the mRNA and protein abundance of KCNQ2 and KCNQ3 in ipsilateral L4/5 DRG tissues obtained from BCP model rats that received intrathecal LV-shEGFR or LV-ZsGreen, performed at 14 days after tumor cells inoculation. ( I and J ) for KCNQ2 ( n = 6–7 rats per group); ( K and L ) for KCNQ3 ( n = 5–7 rats per group). Upper in ( J and L ): Representative blots are shown. ( M - Q ) Electrophysiological analyses of M-currents ( M and O ) and neuronal excitability ( N - Q ) in ipsilateral L4/5 DRG neurons of BCP model rats that received intrathecal LV-shEGFR or LV-ZsGreen, recorded at 14 days after tumor cells inoculation. ( M and N ) Representative traces of M-currents ( M ) and neuronal action potentials ( N ) evoked by a large depolarizing current pulse (1-s, 2-fold AP rheobase) are shown. Scale bar = 100 pA, 300 ms for ( M ), and 20 mV, 100 ms for ( N ). ( O - Q ) Plots of M-current density, frequency of APs, inter-spike intervals (ISI), AP amplitude, after hyperpolarization (AHP) amplitude, AHP 80% duration ( O ), threshold potential ( P ), and rheobase ( Q ) ( n = 20–30 cells from six rats per group). ( R and S ) Assessment of ipsilateral PWT ( R ) and PWL ( S ) for BCP model rats that received intrathecal LV-shEGFR or LV-ZsGreen, performed at 14 days after tumor cells inoculation ( n = 11–12 rats per group). ( T ) Assessment of animal’s locomotor function before and after intrathecal lentivirus administration ( n = 11–12 rats per group). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, unpaired t test for ( A )-( L ) and ( O )-( Q ); two-way ANOVA with Sidak’s post hoc test for ( R )-( T ). See also Fig. S10-S13

Article Snippet: Then, tissues or cultured cells were incubated with the corresponding primary antibody (see Supplementary Table S1) in PBS at 4 °C overnight, which includes rabbit anti-KCNQ2 (1:200, Abcam), rabbit anti-KCNQ3 (1:200, Abcam), rabbit anti-mouse NeuN (1:200, Sigma-Aldrich), mouse anti-pig GFAP (1:200, Cell Signaling Technology), mouse anti-NF200 (1:200, Sigma-Aldrich), mouse anti-CGRP (1:200, Abcam), rabbit anti-HDAC2 (1:200, Abcam), rabbit anti-MeCP2 (1:200, Abcam), goat anti-EGFR antibody (1:200, GeneTex), rabbit anti-Runx1(1:200, Abcam), goat anti-Sin3A (1:200, R&D systems).

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Control

Journal: Cell Communication and Signaling : CCS

Article Title: Involvement of HDAC2-mediated kcnq2/kcnq3 genes transcription repression activated by EREG/EGFR-ERK-Runx1 signaling in bone cancer pain

doi: 10.1186/s12964-024-01797-2

Figure Lengend Snippet: Inhibition of ERK signaling impairs Runx1-dependent upregulation of HDAC2 and HDAC2-mediated transcriptional repression of kcnq2 and kcnq3 genes, reduces neuronal excitability and attenuates pain hypersensitivity in bone cancer pain model rats. ( A and B) Western blot analysis of phosphorylated Runx1 at serine 249 (pRunx1 Ser249 ) ( A ) and Runx1 ( B ) protein abundance in ipsilateral L4/5 DRG tissues obtained from BCP model rats that received intrathecal SCH772984 (a selective ERK inhibitor), performed at 14 days after tumor cells inoculation ( n = 5 rats per group). Upper in ( A ): Representative blots are shown. ( C ) ChIP-qPCR assays for the enrichment of Runx1 in hdac2 gene promoter, in ipsilateral L4/5 DRG tissues obtained from bone cancer pain (BCP) model rats that received intrathecal SCH772984 or vehicle, performed at 14 days after tumor cells inoculation ( n = 4–5 rats per group). ( D ) Western blot analysis of HDAC2 protein abundance in ipsilateral L4/5 DRG tissues obtained from BCP model rats that received intrathecal SCH772984 or vehicle, performed at 14 days after tumor cells inoculation ( n = 6 rats per group). Upper: Representative blots are shown. ( E – H ) RT-qPCR and Western blot analyses of the mRNA and protein abundance of KCNQ2 and KCNQ3 in ipsilateral L4/5 DRG tissues obtained from BCP model rats that received intrathecal SCH772984 or vehicle, performed at 14 days after tumor cells inoculation. ( E and F ) for KCNQ2 ( n = 6 rats per group); ( G and H ) for KCNQ3 ( n = 6–7 rats per group). Upper in ( F and H ): Representative blots are shown. ( I - O ) Electrophysiological analyses of M-currents ( I and M ) and neuronal excitability ( J - O ) in ipsilateral L4/5 DRG neurons of BCP model rats that received intrathecal SCH772984 or vehicle, recorded at 14 days after tumor cells inoculation. ( I and J ) Representative traces of M-currents ( I ) and neuronal action potentials ( J ) evoked by a large depolarizing current pulse (1-s, 2-fold AP rheobase) are shown. Scale bar = 100 pA, 300 ms for ( I ), and 20 mV, 100 ms for ( J ). ( K and L ) Analysis of neuronal firing rate (spikes/second) elicited by a series of 500-ms depolarizing current pulses (in 50-pA steps from 0 to 400 pA). ( K ) Representative traces of evoked action potentials (APs) by 100 pA, 200 pA, and 300 pA depolarizing current pulses are shown. Scale bar = 20 mV, 200 ms. ( M – O ) Plots of M-current density, frequency of APs, inter-spike intervals (ISI), AP amplitude, after hyperpolarization (AHP) amplitude, AHP 80% duration ( M ), threshold potential ( N ), and rheobase ( O ) ( n = 20–30 cells from six rats per group). ( P ) Assessment of ipsilateral PWT for BCP model rats that received intrathecal SCH772984 or vehicle, performed at 14 days after tumor cells inoculation ( n = 10 rats per group). ( Q ) Assessment of animal’s locomotor function before and after intrathecal drug administration ( n = 10 rats per group). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant, unpaired t test for ( A )-( H ) and ( M )-( O ); two-way ANOVA with Sidak’s post hoc test for ( L ), ( P ), and ( Q ). See also Fig. S14-15

Article Snippet: Then, tissues or cultured cells were incubated with the corresponding primary antibody (see Supplementary Table S1) in PBS at 4 °C overnight, which includes rabbit anti-KCNQ2 (1:200, Abcam), rabbit anti-KCNQ3 (1:200, Abcam), rabbit anti-mouse NeuN (1:200, Sigma-Aldrich), mouse anti-pig GFAP (1:200, Cell Signaling Technology), mouse anti-NF200 (1:200, Sigma-Aldrich), mouse anti-CGRP (1:200, Abcam), rabbit anti-HDAC2 (1:200, Abcam), rabbit anti-MeCP2 (1:200, Abcam), goat anti-EGFR antibody (1:200, GeneTex), rabbit anti-Runx1(1:200, Abcam), goat anti-Sin3A (1:200, R&D systems).

Techniques: Inhibition, Western Blot, Quantitative RT-PCR

Journal: Cell Communication and Signaling : CCS

Article Title: Involvement of HDAC2-mediated kcnq2/kcnq3 genes transcription repression activated by EREG/EGFR-ERK-Runx1 signaling in bone cancer pain

doi: 10.1186/s12964-024-01797-2

Figure Lengend Snippet: Schematic summary of HDAC2-mediated kcnq2/kcnq3 genes transcription repression in bone cancer pain. The HDAC2-mediated transcriptional repression of kcnq2 and kcnq3 genes, induced by the activation of EREG/EGFR-ERK-Runx1 signaling, contributes to the sensitization of DRG neurons and the pathogenesis of BCP in rats. Note that HDAC2 needs to form corepressor complex with MeCP2 and Sin3A, and EREG is an upstream signal molecule for HDAC2-mediated gene transcription repression. EREG/EGFR-ERK-Runx1 signaling underlies the HDAC2-mediated gene transcription repression

Article Snippet: Then, tissues or cultured cells were incubated with the corresponding primary antibody (see Supplementary Table S1) in PBS at 4 °C overnight, which includes rabbit anti-KCNQ2 (1:200, Abcam), rabbit anti-KCNQ3 (1:200, Abcam), rabbit anti-mouse NeuN (1:200, Sigma-Aldrich), mouse anti-pig GFAP (1:200, Cell Signaling Technology), mouse anti-NF200 (1:200, Sigma-Aldrich), mouse anti-CGRP (1:200, Abcam), rabbit anti-HDAC2 (1:200, Abcam), rabbit anti-MeCP2 (1:200, Abcam), goat anti-EGFR antibody (1:200, GeneTex), rabbit anti-Runx1(1:200, Abcam), goat anti-Sin3A (1:200, R&D systems).

Techniques: Activation Assay

Journal: British Journal of Pharmacology

Article Title: Functional expression of KCNQ (K v 7) channels in guinea pig bladder smooth muscle and their contribution to spontaneous activity

doi: 10.1111/bph.12210

Figure Lengend Snippet: Guinea pig oligonucleotide sequence of primers used for RT-PCR

Article Snippet: Detrusor preparations were fixed in 4% paraformaldehyde, washed in PBS, blocked in 1% BSA and incubated with primary antibodies (in 0.05% Triton-X 100) to KCNQ subtypes 1–5 (KCNQ1 (APC-022), KCNQ2 (APC-050) KCNQ3 (APC-051) from Alomone, 1:200; KCNQ 4 (sc-50417) and KCNQ5 (sc-50416) from Santa Cruz (1:200)) for 24 h. After washing in PBS to remove excess antibody, tissues were incubated in secondary fluorescent antibodies (Alexa 488 anti-rabbit, 1:200, Invitrogen) for 1 h. Immunolabelled tissues were imaged with confocal microscopy (Nikon C1) mounted on a Nikon e90i upright microscope equipped with a 488 nm argon ion laser and the resulting emission fluorescence collected through appropriate filters to a photomultiplier tube.

Techniques: Sequencing