rabbit anti trek2 antibody  (Alomone Labs)


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    Alomone Labs rabbit anti trek2 antibody
    A , <t>TREK2</t> antibody characterization. Top, GFP fluorescence or interference contrast (IC) shows HEK cells, transfected with plasmids for six rat K2P channels: TREK2, TWIK1, TREK1, TASK3, THIK2, and THIK1 (all subcloned in pCMV-SPORT vector). Bottom, Same fields show immunostaining for TREK2 that was intrinsically very low (GFP alone), increased substantially after transfection with the chimeric TREK2-GFP and with TREK2, but remained very low after transfection with all non-TREK2 K2P channels tested. This antibody therefore selectively recognizes TREK2 and not the other K2P channel tested, including TREK1, structurally the closest related to TREK2. Scale bar, 40 μm. B–D , TREK2 in DRG neurons is selectively expressed in IB4 + neurons. B , Triple fluorescence immunocytochemistry shows TREK2 (blue) to be highly colocalized with IB4 binding (red). RT97 (green) is a marker for neurofilament (against a highly phosphorylated epitope on NF200) that selectively stains DRG somata with A-fibers (i.e., myelinated). TREK2 was only visible in neurofilament-poor (NF-poor, thus C-fiber) neurons, as shown by lack of (green) NF staining in TREK2 + neurons. TREK2 staining is thus in IB4 + DRG neurons with C-fibers. Scale bar, 20 μm. C , D , %intensities refers to cytoplasmic immunointensities expressed as a percentage of maximum staining above background for that marker (see Materials and Methods); gray band(s) show(s) negative staining for TREK2 (
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    Images

    1) Product Images from "TREK2 Expressed Selectively in IB4-Binding C-Fiber Nociceptors Hyperpolarizes Their Membrane Potentials and Limits Spontaneous Pain"

    Article Title: TREK2 Expressed Selectively in IB4-Binding C-Fiber Nociceptors Hyperpolarizes Their Membrane Potentials and Limits Spontaneous Pain

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4528-13.2014

    A , TREK2 antibody characterization. Top, GFP fluorescence or interference contrast (IC) shows HEK cells, transfected with plasmids for six rat K2P channels: TREK2, TWIK1, TREK1, TASK3, THIK2, and THIK1 (all subcloned in pCMV-SPORT vector). Bottom, Same fields show immunostaining for TREK2 that was intrinsically very low (GFP alone), increased substantially after transfection with the chimeric TREK2-GFP and with TREK2, but remained very low after transfection with all non-TREK2 K2P channels tested. This antibody therefore selectively recognizes TREK2 and not the other K2P channel tested, including TREK1, structurally the closest related to TREK2. Scale bar, 40 μm. B–D , TREK2 in DRG neurons is selectively expressed in IB4 + neurons. B , Triple fluorescence immunocytochemistry shows TREK2 (blue) to be highly colocalized with IB4 binding (red). RT97 (green) is a marker for neurofilament (against a highly phosphorylated epitope on NF200) that selectively stains DRG somata with A-fibers (i.e., myelinated). TREK2 was only visible in neurofilament-poor (NF-poor, thus C-fiber) neurons, as shown by lack of (green) NF staining in TREK2 + neurons. TREK2 staining is thus in IB4 + DRG neurons with C-fibers. Scale bar, 20 μm. C , D , %intensities refers to cytoplasmic immunointensities expressed as a percentage of maximum staining above background for that marker (see Materials and Methods); gray band(s) show(s) negative staining for TREK2 (
    Figure Legend Snippet: A , TREK2 antibody characterization. Top, GFP fluorescence or interference contrast (IC) shows HEK cells, transfected with plasmids for six rat K2P channels: TREK2, TWIK1, TREK1, TASK3, THIK2, and THIK1 (all subcloned in pCMV-SPORT vector). Bottom, Same fields show immunostaining for TREK2 that was intrinsically very low (GFP alone), increased substantially after transfection with the chimeric TREK2-GFP and with TREK2, but remained very low after transfection with all non-TREK2 K2P channels tested. This antibody therefore selectively recognizes TREK2 and not the other K2P channel tested, including TREK1, structurally the closest related to TREK2. Scale bar, 40 μm. B–D , TREK2 in DRG neurons is selectively expressed in IB4 + neurons. B , Triple fluorescence immunocytochemistry shows TREK2 (blue) to be highly colocalized with IB4 binding (red). RT97 (green) is a marker for neurofilament (against a highly phosphorylated epitope on NF200) that selectively stains DRG somata with A-fibers (i.e., myelinated). TREK2 was only visible in neurofilament-poor (NF-poor, thus C-fiber) neurons, as shown by lack of (green) NF staining in TREK2 + neurons. TREK2 staining is thus in IB4 + DRG neurons with C-fibers. Scale bar, 20 μm. C , D , %intensities refers to cytoplasmic immunointensities expressed as a percentage of maximum staining above background for that marker (see Materials and Methods); gray band(s) show(s) negative staining for TREK2 (

    Techniques Used: Fluorescence, Transfection, Plasmid Preparation, Immunostaining, Immunocytochemistry, Binding Assay, Marker, Staining, Negative Staining

    Axotomy 7 d earlier decreased Nav1.8 and Nav1.7 as well as TREK2 expression. A , Blinded subjective scoring (0 = no visible staining; 7 = maximum staining intensity) of whole cytoplasmic Nav1.8 immunointensities in L5 DRG sections from 4 normal (N) and 4 axotomized rats ipsilaterally (Ax). Medians plus interquartile ranges are shown. The median decreased significantly after axotomy (Mann–Whitney test). ** p
    Figure Legend Snippet: Axotomy 7 d earlier decreased Nav1.8 and Nav1.7 as well as TREK2 expression. A , Blinded subjective scoring (0 = no visible staining; 7 = maximum staining intensity) of whole cytoplasmic Nav1.8 immunointensities in L5 DRG sections from 4 normal (N) and 4 axotomized rats ipsilaterally (Ax). Medians plus interquartile ranges are shown. The median decreased significantly after axotomy (Mann–Whitney test). ** p

    Techniques Used: Expressing, Staining, MANN-WHITNEY

    In vivo , TREK2 is in small C-fiber nociceptors; their Ems relate to TREK2 levels. A–G , DRG neurons injected in vivo with fluorescent dye after Em recording and sensory receptor property identification (see Materials and Methods). A , Examples (arrows) of fluorescent dye-labeled neurons before (top) and after (bottom) TREK2 ABC immunocytochemistry. TREK2%intensities (see Materials and Methods) were determined at edge-, mid-, and inner- (perinuclear) cytoplasm ( B , inset). The two C-fiber nociceptors (C-Nocis) have (left) a −52 mV Em and strong cytoplasmic, including edge, TREK2 intensity; then (mid-left) a −42 mV Em, with weak edge- but stronger mid- and inner-TREK2. The Aδ nociceptor (mid-right) and Aα/β LTM (far right) were TREK2 − (see Materials and Methods and below). Scale bar, 20 μm. B–G , Cytoplasmic edge- and mid-TREK2%intensities of ≥25% (of maximum cytoplasmic staining; see Materials and Methods) were deemed positive. The negative staining (
    Figure Legend Snippet: In vivo , TREK2 is in small C-fiber nociceptors; their Ems relate to TREK2 levels. A–G , DRG neurons injected in vivo with fluorescent dye after Em recording and sensory receptor property identification (see Materials and Methods). A , Examples (arrows) of fluorescent dye-labeled neurons before (top) and after (bottom) TREK2 ABC immunocytochemistry. TREK2%intensities (see Materials and Methods) were determined at edge-, mid-, and inner- (perinuclear) cytoplasm ( B , inset). The two C-fiber nociceptors (C-Nocis) have (left) a −52 mV Em and strong cytoplasmic, including edge, TREK2 intensity; then (mid-left) a −42 mV Em, with weak edge- but stronger mid- and inner-TREK2. The Aδ nociceptor (mid-right) and Aα/β LTM (far right) were TREK2 − (see Materials and Methods and below). Scale bar, 20 μm. B–G , Cytoplasmic edge- and mid-TREK2%intensities of ≥25% (of maximum cytoplasmic staining; see Materials and Methods) were deemed positive. The negative staining (

    Techniques Used: In Vivo, Injection, Labeling, Immunocytochemistry, Staining, Negative Staining

    TREK2 knockdown in vivo decreased SFL 1 d after CFA. Ipsilateral SFL in rats 1 d after an intradermal plantar CFA injection in one hind foot in the same region as an intradermal plantar injection of either TREK2 siRNA or scrambled siRNA given 3 d earlier. Numbers in B and C (superscripts) and D and E (beside data points) relate to rats from which tissue in the lanes of A was obtained; these numbers are included to enable comparison of TREK2 protein expression with other data from the same rats. A , TREK2 Western blot of skin tissue, at the site of scr (1, 2) or siRNA (3, 4) injection 4 d previously, and 1 d after either saline (1, 3) or CFA injection (2, 4) at the same site. Top, The lower TREK2 band (MW ∼50 kDa, the band that we and others find in DRG tissues) was almost eliminated by siRNA, not scr, treatment, regardless of whether rats were injected with CFA or saline. The upper band was not reduced after siRNA. The MWs of these bands are consistent with them being two of the three known rat TREK2 isoforms (see Results and Discussion). Bottom, α-Tubulin loading controls. B , TREK2 knockdown by TREK2 siRNA in nerve fibers and epidermis (Ep) in plantar skin. Left, after scr siRNA injection. Right, after TREK2 siRNA injection. Both treatments were followed by CFA injection. Tissue was taken 4 d after scr/siRNA treatment (1 d after CFA). Triple fluorescence staining for β-tubulin III (blue, to stain nerve fibers), IB4 binding (green), and TREK2 (red). White dashed lines indicate junctions between epidermis (above) and dermis (below). In the merged images, colocalization of all three markers shows as white or very pale (bottom left image). After siRNA, red TREK2 staining in epidermis and in groups of nerve fibers (shown as blue and/or green) was reduced, showing substantial knockdown in vivo of TREK2 near the siRNA injection site. Scale bar, 25 μm. C , TREK2 knockdown by local TREK2 siRNA in the nerve innervating plantar skin. This knockdown, seen 5–10 mm from the siRNA injection site, occurred whether rats were subsequently treated with saline or CFA. There was decreased fiber TREK2 immunostaining (red) in TREK2 siRNA, not scr siRNA, treated rats, so that IB4 + fibers (green) showed little/no TREK2 (red). Scale bar, 50 μm. D , E , SFL after CFA increased after TREK2 siRNA compared with scr control. Ipsilateral SFLd ( D ) or SFLn ( E ) after siRNA C compared with scr treatment did not increase before saline injection (day 3, columns 1 and 2) or after saline injection (day 4, columns 3 and 4), or before CFA injection (day 3, columns 5 and 6). However, after CFA, it caused increased SFLd ( D ) and SFLn ( E ) in both scr ( p
    Figure Legend Snippet: TREK2 knockdown in vivo decreased SFL 1 d after CFA. Ipsilateral SFL in rats 1 d after an intradermal plantar CFA injection in one hind foot in the same region as an intradermal plantar injection of either TREK2 siRNA or scrambled siRNA given 3 d earlier. Numbers in B and C (superscripts) and D and E (beside data points) relate to rats from which tissue in the lanes of A was obtained; these numbers are included to enable comparison of TREK2 protein expression with other data from the same rats. A , TREK2 Western blot of skin tissue, at the site of scr (1, 2) or siRNA (3, 4) injection 4 d previously, and 1 d after either saline (1, 3) or CFA injection (2, 4) at the same site. Top, The lower TREK2 band (MW ∼50 kDa, the band that we and others find in DRG tissues) was almost eliminated by siRNA, not scr, treatment, regardless of whether rats were injected with CFA or saline. The upper band was not reduced after siRNA. The MWs of these bands are consistent with them being two of the three known rat TREK2 isoforms (see Results and Discussion). Bottom, α-Tubulin loading controls. B , TREK2 knockdown by TREK2 siRNA in nerve fibers and epidermis (Ep) in plantar skin. Left, after scr siRNA injection. Right, after TREK2 siRNA injection. Both treatments were followed by CFA injection. Tissue was taken 4 d after scr/siRNA treatment (1 d after CFA). Triple fluorescence staining for β-tubulin III (blue, to stain nerve fibers), IB4 binding (green), and TREK2 (red). White dashed lines indicate junctions between epidermis (above) and dermis (below). In the merged images, colocalization of all three markers shows as white or very pale (bottom left image). After siRNA, red TREK2 staining in epidermis and in groups of nerve fibers (shown as blue and/or green) was reduced, showing substantial knockdown in vivo of TREK2 near the siRNA injection site. Scale bar, 25 μm. C , TREK2 knockdown by local TREK2 siRNA in the nerve innervating plantar skin. This knockdown, seen 5–10 mm from the siRNA injection site, occurred whether rats were subsequently treated with saline or CFA. There was decreased fiber TREK2 immunostaining (red) in TREK2 siRNA, not scr siRNA, treated rats, so that IB4 + fibers (green) showed little/no TREK2 (red). Scale bar, 50 μm. D , E , SFL after CFA increased after TREK2 siRNA compared with scr control. Ipsilateral SFLd ( D ) or SFLn ( E ) after siRNA C compared with scr treatment did not increase before saline injection (day 3, columns 1 and 2) or after saline injection (day 4, columns 3 and 4), or before CFA injection (day 3, columns 5 and 6). However, after CFA, it caused increased SFLd ( D ) and SFLn ( E ) in both scr ( p

    Techniques Used: In Vivo, Injection, Expressing, Western Blot, Fluorescence, Staining, Binding Assay, Immunostaining

    A , SF rate was related to Em in C-neurons in neuropathic pain models. SF rate in intact L4 C-nociceptors was significantly linearly correlated with Em 7 d after SNA and mSNA surgery (see Materials and Methods and Results). B , C , SFL 1 d after CFA was related to ipsilateral TREK2 levels. In ipsilateral L5 DRG small neurons in 5 rats 1 d after CFA-induced cutaneous inflammation, there were significant linear correlations of ( B ) SFLd and ( C ) number of SFLn with median ipsilateral L5 small DRG neuron TREK2 pixel densities for each rat, whether in entire cytoplasm, or edge, mid, or inner cytoplasmic regions. Thus, SFL after CFA in individual rats was related to ipsilateral TREK2 levels in small DRG neurons.
    Figure Legend Snippet: A , SF rate was related to Em in C-neurons in neuropathic pain models. SF rate in intact L4 C-nociceptors was significantly linearly correlated with Em 7 d after SNA and mSNA surgery (see Materials and Methods and Results). B , C , SFL 1 d after CFA was related to ipsilateral TREK2 levels. In ipsilateral L5 DRG small neurons in 5 rats 1 d after CFA-induced cutaneous inflammation, there were significant linear correlations of ( B ) SFLd and ( C ) number of SFLn with median ipsilateral L5 small DRG neuron TREK2 pixel densities for each rat, whether in entire cytoplasm, or edge, mid, or inner cytoplasmic regions. Thus, SFL after CFA in individual rats was related to ipsilateral TREK2 levels in small DRG neurons.

    Techniques Used:

    2) Product Images from "TREK2 Expressed Selectively in IB4-Binding C-Fiber Nociceptors Hyperpolarizes Their Membrane Potentials and Limits Spontaneous Pain"

    Article Title: TREK2 Expressed Selectively in IB4-Binding C-Fiber Nociceptors Hyperpolarizes Their Membrane Potentials and Limits Spontaneous Pain

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4528-13.2014

    A , TREK2 antibody characterization. Top, GFP fluorescence or interference contrast (IC) shows HEK cells, transfected with plasmids for six rat K2P channels: TREK2, TWIK1, TREK1, TASK3, THIK2, and THIK1 (all subcloned in pCMV-SPORT vector). Bottom, Same fields show immunostaining for TREK2 that was intrinsically very low (GFP alone), increased substantially after transfection with the chimeric TREK2-GFP and with TREK2, but remained very low after transfection with all non-TREK2 K2P channels tested. This antibody therefore selectively recognizes TREK2 and not the other K2P channel tested, including TREK1, structurally the closest related to TREK2. Scale bar, 40 μm. B–D , TREK2 in DRG neurons is selectively expressed in IB4 + neurons. B , Triple fluorescence immunocytochemistry shows TREK2 (blue) to be highly colocalized with IB4 binding (red). RT97 (green) is a marker for neurofilament (against a highly phosphorylated epitope on NF200) that selectively stains DRG somata with A-fibers (i.e., myelinated). TREK2 was only visible in neurofilament-poor (NF-poor, thus C-fiber) neurons, as shown by lack of (green) NF staining in TREK2 + neurons. TREK2 staining is thus in IB4 + DRG neurons with C-fibers. Scale bar, 20 μm. C , D , %intensities refers to cytoplasmic immunointensities expressed as a percentage of maximum staining above background for that marker (see Materials and Methods); gray band(s) show(s) negative staining for TREK2 (
    Figure Legend Snippet: A , TREK2 antibody characterization. Top, GFP fluorescence or interference contrast (IC) shows HEK cells, transfected with plasmids for six rat K2P channels: TREK2, TWIK1, TREK1, TASK3, THIK2, and THIK1 (all subcloned in pCMV-SPORT vector). Bottom, Same fields show immunostaining for TREK2 that was intrinsically very low (GFP alone), increased substantially after transfection with the chimeric TREK2-GFP and with TREK2, but remained very low after transfection with all non-TREK2 K2P channels tested. This antibody therefore selectively recognizes TREK2 and not the other K2P channel tested, including TREK1, structurally the closest related to TREK2. Scale bar, 40 μm. B–D , TREK2 in DRG neurons is selectively expressed in IB4 + neurons. B , Triple fluorescence immunocytochemistry shows TREK2 (blue) to be highly colocalized with IB4 binding (red). RT97 (green) is a marker for neurofilament (against a highly phosphorylated epitope on NF200) that selectively stains DRG somata with A-fibers (i.e., myelinated). TREK2 was only visible in neurofilament-poor (NF-poor, thus C-fiber) neurons, as shown by lack of (green) NF staining in TREK2 + neurons. TREK2 staining is thus in IB4 + DRG neurons with C-fibers. Scale bar, 20 μm. C , D , %intensities refers to cytoplasmic immunointensities expressed as a percentage of maximum staining above background for that marker (see Materials and Methods); gray band(s) show(s) negative staining for TREK2 (

    Techniques Used: Fluorescence, Transfection, Plasmid Preparation, Immunostaining, Immunocytochemistry, Binding Assay, Marker, Staining, Negative Staining

    Axotomy 7 d earlier decreased Nav1.8 and Nav1.7 as well as TREK2 expression. A , Blinded subjective scoring (0 = no visible staining; 7 = maximum staining intensity) of whole cytoplasmic Nav1.8 immunointensities in L5 DRG sections from 4 normal (N) and 4 axotomized rats ipsilaterally (Ax). Medians plus interquartile ranges are shown. The median decreased significantly after axotomy (Mann–Whitney test). ** p
    Figure Legend Snippet: Axotomy 7 d earlier decreased Nav1.8 and Nav1.7 as well as TREK2 expression. A , Blinded subjective scoring (0 = no visible staining; 7 = maximum staining intensity) of whole cytoplasmic Nav1.8 immunointensities in L5 DRG sections from 4 normal (N) and 4 axotomized rats ipsilaterally (Ax). Medians plus interquartile ranges are shown. The median decreased significantly after axotomy (Mann–Whitney test). ** p

    Techniques Used: Expressing, Staining, MANN-WHITNEY

    In vivo , TREK2 is in small C-fiber nociceptors; their Ems relate to TREK2 levels. A–G , DRG neurons injected in vivo with fluorescent dye after Em recording and sensory receptor property identification (see Materials and Methods). A , Examples (arrows) of fluorescent dye-labeled neurons before (top) and after (bottom) TREK2 ABC immunocytochemistry. TREK2%intensities (see Materials and Methods) were determined at edge-, mid-, and inner- (perinuclear) cytoplasm ( B , inset). The two C-fiber nociceptors (C-Nocis) have (left) a −52 mV Em and strong cytoplasmic, including edge, TREK2 intensity; then (mid-left) a −42 mV Em, with weak edge- but stronger mid- and inner-TREK2. The Aδ nociceptor (mid-right) and Aα/β LTM (far right) were TREK2 − (see Materials and Methods and below). Scale bar, 20 μm. B–G , Cytoplasmic edge- and mid-TREK2%intensities of ≥25% (of maximum cytoplasmic staining; see Materials and Methods) were deemed positive. The negative staining (
    Figure Legend Snippet: In vivo , TREK2 is in small C-fiber nociceptors; their Ems relate to TREK2 levels. A–G , DRG neurons injected in vivo with fluorescent dye after Em recording and sensory receptor property identification (see Materials and Methods). A , Examples (arrows) of fluorescent dye-labeled neurons before (top) and after (bottom) TREK2 ABC immunocytochemistry. TREK2%intensities (see Materials and Methods) were determined at edge-, mid-, and inner- (perinuclear) cytoplasm ( B , inset). The two C-fiber nociceptors (C-Nocis) have (left) a −52 mV Em and strong cytoplasmic, including edge, TREK2 intensity; then (mid-left) a −42 mV Em, with weak edge- but stronger mid- and inner-TREK2. The Aδ nociceptor (mid-right) and Aα/β LTM (far right) were TREK2 − (see Materials and Methods and below). Scale bar, 20 μm. B–G , Cytoplasmic edge- and mid-TREK2%intensities of ≥25% (of maximum cytoplasmic staining; see Materials and Methods) were deemed positive. The negative staining (

    Techniques Used: In Vivo, Injection, Labeling, Immunocytochemistry, Staining, Negative Staining

    TREK2 knockdown in vivo decreased SFL 1 d after CFA. Ipsilateral SFL in rats 1 d after an intradermal plantar CFA injection in one hind foot in the same region as an intradermal plantar injection of either TREK2 siRNA or scrambled siRNA given 3 d earlier. Numbers in B and C (superscripts) and D and E (beside data points) relate to rats from which tissue in the lanes of A was obtained; these numbers are included to enable comparison of TREK2 protein expression with other data from the same rats. A , TREK2 Western blot of skin tissue, at the site of scr (1, 2) or siRNA (3, 4) injection 4 d previously, and 1 d after either saline (1, 3) or CFA injection (2, 4) at the same site. Top, The lower TREK2 band (MW ∼50 kDa, the band that we and others find in DRG tissues) was almost eliminated by siRNA, not scr, treatment, regardless of whether rats were injected with CFA or saline. The upper band was not reduced after siRNA. The MWs of these bands are consistent with them being two of the three known rat TREK2 isoforms (see Results and Discussion). Bottom, α-Tubulin loading controls. B , TREK2 knockdown by TREK2 siRNA in nerve fibers and epidermis (Ep) in plantar skin. Left, after scr siRNA injection. Right, after TREK2 siRNA injection. Both treatments were followed by CFA injection. Tissue was taken 4 d after scr/siRNA treatment (1 d after CFA). Triple fluorescence staining for β-tubulin III (blue, to stain nerve fibers), IB4 binding (green), and TREK2 (red). White dashed lines indicate junctions between epidermis (above) and dermis (below). In the merged images, colocalization of all three markers shows as white or very pale (bottom left image). After siRNA, red TREK2 staining in epidermis and in groups of nerve fibers (shown as blue and/or green) was reduced, showing substantial knockdown in vivo of TREK2 near the siRNA injection site. Scale bar, 25 μm. C , TREK2 knockdown by local TREK2 siRNA in the nerve innervating plantar skin. This knockdown, seen 5–10 mm from the siRNA injection site, occurred whether rats were subsequently treated with saline or CFA. There was decreased fiber TREK2 immunostaining (red) in TREK2 siRNA, not scr siRNA, treated rats, so that IB4 + fibers (green) showed little/no TREK2 (red). Scale bar, 50 μm. D , E , SFL after CFA increased after TREK2 siRNA compared with scr control. Ipsilateral SFLd ( D ) or SFLn ( E ) after siRNA C compared with scr treatment did not increase before saline injection (day 3, columns 1 and 2) or after saline injection (day 4, columns 3 and 4), or before CFA injection (day 3, columns 5 and 6). However, after CFA, it caused increased SFLd ( D ) and SFLn ( E ) in both scr ( p
    Figure Legend Snippet: TREK2 knockdown in vivo decreased SFL 1 d after CFA. Ipsilateral SFL in rats 1 d after an intradermal plantar CFA injection in one hind foot in the same region as an intradermal plantar injection of either TREK2 siRNA or scrambled siRNA given 3 d earlier. Numbers in B and C (superscripts) and D and E (beside data points) relate to rats from which tissue in the lanes of A was obtained; these numbers are included to enable comparison of TREK2 protein expression with other data from the same rats. A , TREK2 Western blot of skin tissue, at the site of scr (1, 2) or siRNA (3, 4) injection 4 d previously, and 1 d after either saline (1, 3) or CFA injection (2, 4) at the same site. Top, The lower TREK2 band (MW ∼50 kDa, the band that we and others find in DRG tissues) was almost eliminated by siRNA, not scr, treatment, regardless of whether rats were injected with CFA or saline. The upper band was not reduced after siRNA. The MWs of these bands are consistent with them being two of the three known rat TREK2 isoforms (see Results and Discussion). Bottom, α-Tubulin loading controls. B , TREK2 knockdown by TREK2 siRNA in nerve fibers and epidermis (Ep) in plantar skin. Left, after scr siRNA injection. Right, after TREK2 siRNA injection. Both treatments were followed by CFA injection. Tissue was taken 4 d after scr/siRNA treatment (1 d after CFA). Triple fluorescence staining for β-tubulin III (blue, to stain nerve fibers), IB4 binding (green), and TREK2 (red). White dashed lines indicate junctions between epidermis (above) and dermis (below). In the merged images, colocalization of all three markers shows as white or very pale (bottom left image). After siRNA, red TREK2 staining in epidermis and in groups of nerve fibers (shown as blue and/or green) was reduced, showing substantial knockdown in vivo of TREK2 near the siRNA injection site. Scale bar, 25 μm. C , TREK2 knockdown by local TREK2 siRNA in the nerve innervating plantar skin. This knockdown, seen 5–10 mm from the siRNA injection site, occurred whether rats were subsequently treated with saline or CFA. There was decreased fiber TREK2 immunostaining (red) in TREK2 siRNA, not scr siRNA, treated rats, so that IB4 + fibers (green) showed little/no TREK2 (red). Scale bar, 50 μm. D , E , SFL after CFA increased after TREK2 siRNA compared with scr control. Ipsilateral SFLd ( D ) or SFLn ( E ) after siRNA C compared with scr treatment did not increase before saline injection (day 3, columns 1 and 2) or after saline injection (day 4, columns 3 and 4), or before CFA injection (day 3, columns 5 and 6). However, after CFA, it caused increased SFLd ( D ) and SFLn ( E ) in both scr ( p

    Techniques Used: In Vivo, Injection, Expressing, Western Blot, Fluorescence, Staining, Binding Assay, Immunostaining

    A , SF rate was related to Em in C-neurons in neuropathic pain models. SF rate in intact L4 C-nociceptors was significantly linearly correlated with Em 7 d after SNA and mSNA surgery (see Materials and Methods and Results). B , C , SFL 1 d after CFA was related to ipsilateral TREK2 levels. In ipsilateral L5 DRG small neurons in 5 rats 1 d after CFA-induced cutaneous inflammation, there were significant linear correlations of ( B ) SFLd and ( C ) number of SFLn with median ipsilateral L5 small DRG neuron TREK2 pixel densities for each rat, whether in entire cytoplasm, or edge, mid, or inner cytoplasmic regions. Thus, SFL after CFA in individual rats was related to ipsilateral TREK2 levels in small DRG neurons.
    Figure Legend Snippet: A , SF rate was related to Em in C-neurons in neuropathic pain models. SF rate in intact L4 C-nociceptors was significantly linearly correlated with Em 7 d after SNA and mSNA surgery (see Materials and Methods and Results). B , C , SFL 1 d after CFA was related to ipsilateral TREK2 levels. In ipsilateral L5 DRG small neurons in 5 rats 1 d after CFA-induced cutaneous inflammation, there were significant linear correlations of ( B ) SFLd and ( C ) number of SFLn with median ipsilateral L5 small DRG neuron TREK2 pixel densities for each rat, whether in entire cytoplasm, or edge, mid, or inner cytoplasmic regions. Thus, SFL after CFA in individual rats was related to ipsilateral TREK2 levels in small DRG neurons.

    Techniques Used:

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  • 93
    Alomone Labs rabbit anti trek2 antibody
    A , <t>TREK2</t> antibody characterization. Top, GFP fluorescence or interference contrast (IC) shows HEK cells, transfected with plasmids for six rat K2P channels: TREK2, TWIK1, TREK1, TASK3, THIK2, and THIK1 (all subcloned in pCMV-SPORT vector). Bottom, Same fields show immunostaining for TREK2 that was intrinsically very low (GFP alone), increased substantially after transfection with the chimeric TREK2-GFP and with TREK2, but remained very low after transfection with all non-TREK2 K2P channels tested. This antibody therefore selectively recognizes TREK2 and not the other K2P channel tested, including TREK1, structurally the closest related to TREK2. Scale bar, 40 μm. B–D , TREK2 in DRG neurons is selectively expressed in IB4 + neurons. B , Triple fluorescence immunocytochemistry shows TREK2 (blue) to be highly colocalized with IB4 binding (red). RT97 (green) is a marker for neurofilament (against a highly phosphorylated epitope on NF200) that selectively stains DRG somata with A-fibers (i.e., myelinated). TREK2 was only visible in neurofilament-poor (NF-poor, thus C-fiber) neurons, as shown by lack of (green) NF staining in TREK2 + neurons. TREK2 staining is thus in IB4 + DRG neurons with C-fibers. Scale bar, 20 μm. C , D , %intensities refers to cytoplasmic immunointensities expressed as a percentage of maximum staining above background for that marker (see Materials and Methods); gray band(s) show(s) negative staining for TREK2 (
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    A , TREK2 antibody characterization. Top, GFP fluorescence or interference contrast (IC) shows HEK cells, transfected with plasmids for six rat K2P channels: TREK2, TWIK1, TREK1, TASK3, THIK2, and THIK1 (all subcloned in pCMV-SPORT vector). Bottom, Same fields show immunostaining for TREK2 that was intrinsically very low (GFP alone), increased substantially after transfection with the chimeric TREK2-GFP and with TREK2, but remained very low after transfection with all non-TREK2 K2P channels tested. This antibody therefore selectively recognizes TREK2 and not the other K2P channel tested, including TREK1, structurally the closest related to TREK2. Scale bar, 40 μm. B–D , TREK2 in DRG neurons is selectively expressed in IB4 + neurons. B , Triple fluorescence immunocytochemistry shows TREK2 (blue) to be highly colocalized with IB4 binding (red). RT97 (green) is a marker for neurofilament (against a highly phosphorylated epitope on NF200) that selectively stains DRG somata with A-fibers (i.e., myelinated). TREK2 was only visible in neurofilament-poor (NF-poor, thus C-fiber) neurons, as shown by lack of (green) NF staining in TREK2 + neurons. TREK2 staining is thus in IB4 + DRG neurons with C-fibers. Scale bar, 20 μm. C , D , %intensities refers to cytoplasmic immunointensities expressed as a percentage of maximum staining above background for that marker (see Materials and Methods); gray band(s) show(s) negative staining for TREK2 (

    Journal: The Journal of Neuroscience

    Article Title: TREK2 Expressed Selectively in IB4-Binding C-Fiber Nociceptors Hyperpolarizes Their Membrane Potentials and Limits Spontaneous Pain

    doi: 10.1523/JNEUROSCI.4528-13.2014

    Figure Lengend Snippet: A , TREK2 antibody characterization. Top, GFP fluorescence or interference contrast (IC) shows HEK cells, transfected with plasmids for six rat K2P channels: TREK2, TWIK1, TREK1, TASK3, THIK2, and THIK1 (all subcloned in pCMV-SPORT vector). Bottom, Same fields show immunostaining for TREK2 that was intrinsically very low (GFP alone), increased substantially after transfection with the chimeric TREK2-GFP and with TREK2, but remained very low after transfection with all non-TREK2 K2P channels tested. This antibody therefore selectively recognizes TREK2 and not the other K2P channel tested, including TREK1, structurally the closest related to TREK2. Scale bar, 40 μm. B–D , TREK2 in DRG neurons is selectively expressed in IB4 + neurons. B , Triple fluorescence immunocytochemistry shows TREK2 (blue) to be highly colocalized with IB4 binding (red). RT97 (green) is a marker for neurofilament (against a highly phosphorylated epitope on NF200) that selectively stains DRG somata with A-fibers (i.e., myelinated). TREK2 was only visible in neurofilament-poor (NF-poor, thus C-fiber) neurons, as shown by lack of (green) NF staining in TREK2 + neurons. TREK2 staining is thus in IB4 + DRG neurons with C-fibers. Scale bar, 20 μm. C , D , %intensities refers to cytoplasmic immunointensities expressed as a percentage of maximum staining above background for that marker (see Materials and Methods); gray band(s) show(s) negative staining for TREK2 (

    Article Snippet: Three days after transfection, the HEK cell cultures expressing the different K2P channels were washed with PBS, fixed with 4% paraformaldehyde in 4.2% sucrose (in PBS, pH 7.2) for 10 min at room temperature, rinsed with PBS, and processed for TREK2 immunocytochemistry with the rabbit anti-TREK2 antibody from Alomone Labs as described above.

    Techniques: Fluorescence, Transfection, Plasmid Preparation, Immunostaining, Immunocytochemistry, Binding Assay, Marker, Staining, Negative Staining

    Axotomy 7 d earlier decreased Nav1.8 and Nav1.7 as well as TREK2 expression. A , Blinded subjective scoring (0 = no visible staining; 7 = maximum staining intensity) of whole cytoplasmic Nav1.8 immunointensities in L5 DRG sections from 4 normal (N) and 4 axotomized rats ipsilaterally (Ax). Medians plus interquartile ranges are shown. The median decreased significantly after axotomy (Mann–Whitney test). ** p

    Journal: The Journal of Neuroscience

    Article Title: TREK2 Expressed Selectively in IB4-Binding C-Fiber Nociceptors Hyperpolarizes Their Membrane Potentials and Limits Spontaneous Pain

    doi: 10.1523/JNEUROSCI.4528-13.2014

    Figure Lengend Snippet: Axotomy 7 d earlier decreased Nav1.8 and Nav1.7 as well as TREK2 expression. A , Blinded subjective scoring (0 = no visible staining; 7 = maximum staining intensity) of whole cytoplasmic Nav1.8 immunointensities in L5 DRG sections from 4 normal (N) and 4 axotomized rats ipsilaterally (Ax). Medians plus interquartile ranges are shown. The median decreased significantly after axotomy (Mann–Whitney test). ** p

    Article Snippet: Three days after transfection, the HEK cell cultures expressing the different K2P channels were washed with PBS, fixed with 4% paraformaldehyde in 4.2% sucrose (in PBS, pH 7.2) for 10 min at room temperature, rinsed with PBS, and processed for TREK2 immunocytochemistry with the rabbit anti-TREK2 antibody from Alomone Labs as described above.

    Techniques: Expressing, Staining, MANN-WHITNEY

    In vivo , TREK2 is in small C-fiber nociceptors; their Ems relate to TREK2 levels. A–G , DRG neurons injected in vivo with fluorescent dye after Em recording and sensory receptor property identification (see Materials and Methods). A , Examples (arrows) of fluorescent dye-labeled neurons before (top) and after (bottom) TREK2 ABC immunocytochemistry. TREK2%intensities (see Materials and Methods) were determined at edge-, mid-, and inner- (perinuclear) cytoplasm ( B , inset). The two C-fiber nociceptors (C-Nocis) have (left) a −52 mV Em and strong cytoplasmic, including edge, TREK2 intensity; then (mid-left) a −42 mV Em, with weak edge- but stronger mid- and inner-TREK2. The Aδ nociceptor (mid-right) and Aα/β LTM (far right) were TREK2 − (see Materials and Methods and below). Scale bar, 20 μm. B–G , Cytoplasmic edge- and mid-TREK2%intensities of ≥25% (of maximum cytoplasmic staining; see Materials and Methods) were deemed positive. The negative staining (

    Journal: The Journal of Neuroscience

    Article Title: TREK2 Expressed Selectively in IB4-Binding C-Fiber Nociceptors Hyperpolarizes Their Membrane Potentials and Limits Spontaneous Pain

    doi: 10.1523/JNEUROSCI.4528-13.2014

    Figure Lengend Snippet: In vivo , TREK2 is in small C-fiber nociceptors; their Ems relate to TREK2 levels. A–G , DRG neurons injected in vivo with fluorescent dye after Em recording and sensory receptor property identification (see Materials and Methods). A , Examples (arrows) of fluorescent dye-labeled neurons before (top) and after (bottom) TREK2 ABC immunocytochemistry. TREK2%intensities (see Materials and Methods) were determined at edge-, mid-, and inner- (perinuclear) cytoplasm ( B , inset). The two C-fiber nociceptors (C-Nocis) have (left) a −52 mV Em and strong cytoplasmic, including edge, TREK2 intensity; then (mid-left) a −42 mV Em, with weak edge- but stronger mid- and inner-TREK2. The Aδ nociceptor (mid-right) and Aα/β LTM (far right) were TREK2 − (see Materials and Methods and below). Scale bar, 20 μm. B–G , Cytoplasmic edge- and mid-TREK2%intensities of ≥25% (of maximum cytoplasmic staining; see Materials and Methods) were deemed positive. The negative staining (

    Article Snippet: Three days after transfection, the HEK cell cultures expressing the different K2P channels were washed with PBS, fixed with 4% paraformaldehyde in 4.2% sucrose (in PBS, pH 7.2) for 10 min at room temperature, rinsed with PBS, and processed for TREK2 immunocytochemistry with the rabbit anti-TREK2 antibody from Alomone Labs as described above.

    Techniques: In Vivo, Injection, Labeling, Immunocytochemistry, Staining, Negative Staining

    TREK2 knockdown in vivo decreased SFL 1 d after CFA. Ipsilateral SFL in rats 1 d after an intradermal plantar CFA injection in one hind foot in the same region as an intradermal plantar injection of either TREK2 siRNA or scrambled siRNA given 3 d earlier. Numbers in B and C (superscripts) and D and E (beside data points) relate to rats from which tissue in the lanes of A was obtained; these numbers are included to enable comparison of TREK2 protein expression with other data from the same rats. A , TREK2 Western blot of skin tissue, at the site of scr (1, 2) or siRNA (3, 4) injection 4 d previously, and 1 d after either saline (1, 3) or CFA injection (2, 4) at the same site. Top, The lower TREK2 band (MW ∼50 kDa, the band that we and others find in DRG tissues) was almost eliminated by siRNA, not scr, treatment, regardless of whether rats were injected with CFA or saline. The upper band was not reduced after siRNA. The MWs of these bands are consistent with them being two of the three known rat TREK2 isoforms (see Results and Discussion). Bottom, α-Tubulin loading controls. B , TREK2 knockdown by TREK2 siRNA in nerve fibers and epidermis (Ep) in plantar skin. Left, after scr siRNA injection. Right, after TREK2 siRNA injection. Both treatments were followed by CFA injection. Tissue was taken 4 d after scr/siRNA treatment (1 d after CFA). Triple fluorescence staining for β-tubulin III (blue, to stain nerve fibers), IB4 binding (green), and TREK2 (red). White dashed lines indicate junctions between epidermis (above) and dermis (below). In the merged images, colocalization of all three markers shows as white or very pale (bottom left image). After siRNA, red TREK2 staining in epidermis and in groups of nerve fibers (shown as blue and/or green) was reduced, showing substantial knockdown in vivo of TREK2 near the siRNA injection site. Scale bar, 25 μm. C , TREK2 knockdown by local TREK2 siRNA in the nerve innervating plantar skin. This knockdown, seen 5–10 mm from the siRNA injection site, occurred whether rats were subsequently treated with saline or CFA. There was decreased fiber TREK2 immunostaining (red) in TREK2 siRNA, not scr siRNA, treated rats, so that IB4 + fibers (green) showed little/no TREK2 (red). Scale bar, 50 μm. D , E , SFL after CFA increased after TREK2 siRNA compared with scr control. Ipsilateral SFLd ( D ) or SFLn ( E ) after siRNA C compared with scr treatment did not increase before saline injection (day 3, columns 1 and 2) or after saline injection (day 4, columns 3 and 4), or before CFA injection (day 3, columns 5 and 6). However, after CFA, it caused increased SFLd ( D ) and SFLn ( E ) in both scr ( p

    Journal: The Journal of Neuroscience

    Article Title: TREK2 Expressed Selectively in IB4-Binding C-Fiber Nociceptors Hyperpolarizes Their Membrane Potentials and Limits Spontaneous Pain

    doi: 10.1523/JNEUROSCI.4528-13.2014

    Figure Lengend Snippet: TREK2 knockdown in vivo decreased SFL 1 d after CFA. Ipsilateral SFL in rats 1 d after an intradermal plantar CFA injection in one hind foot in the same region as an intradermal plantar injection of either TREK2 siRNA or scrambled siRNA given 3 d earlier. Numbers in B and C (superscripts) and D and E (beside data points) relate to rats from which tissue in the lanes of A was obtained; these numbers are included to enable comparison of TREK2 protein expression with other data from the same rats. A , TREK2 Western blot of skin tissue, at the site of scr (1, 2) or siRNA (3, 4) injection 4 d previously, and 1 d after either saline (1, 3) or CFA injection (2, 4) at the same site. Top, The lower TREK2 band (MW ∼50 kDa, the band that we and others find in DRG tissues) was almost eliminated by siRNA, not scr, treatment, regardless of whether rats were injected with CFA or saline. The upper band was not reduced after siRNA. The MWs of these bands are consistent with them being two of the three known rat TREK2 isoforms (see Results and Discussion). Bottom, α-Tubulin loading controls. B , TREK2 knockdown by TREK2 siRNA in nerve fibers and epidermis (Ep) in plantar skin. Left, after scr siRNA injection. Right, after TREK2 siRNA injection. Both treatments were followed by CFA injection. Tissue was taken 4 d after scr/siRNA treatment (1 d after CFA). Triple fluorescence staining for β-tubulin III (blue, to stain nerve fibers), IB4 binding (green), and TREK2 (red). White dashed lines indicate junctions between epidermis (above) and dermis (below). In the merged images, colocalization of all three markers shows as white or very pale (bottom left image). After siRNA, red TREK2 staining in epidermis and in groups of nerve fibers (shown as blue and/or green) was reduced, showing substantial knockdown in vivo of TREK2 near the siRNA injection site. Scale bar, 25 μm. C , TREK2 knockdown by local TREK2 siRNA in the nerve innervating plantar skin. This knockdown, seen 5–10 mm from the siRNA injection site, occurred whether rats were subsequently treated with saline or CFA. There was decreased fiber TREK2 immunostaining (red) in TREK2 siRNA, not scr siRNA, treated rats, so that IB4 + fibers (green) showed little/no TREK2 (red). Scale bar, 50 μm. D , E , SFL after CFA increased after TREK2 siRNA compared with scr control. Ipsilateral SFLd ( D ) or SFLn ( E ) after siRNA C compared with scr treatment did not increase before saline injection (day 3, columns 1 and 2) or after saline injection (day 4, columns 3 and 4), or before CFA injection (day 3, columns 5 and 6). However, after CFA, it caused increased SFLd ( D ) and SFLn ( E ) in both scr ( p

    Article Snippet: Three days after transfection, the HEK cell cultures expressing the different K2P channels were washed with PBS, fixed with 4% paraformaldehyde in 4.2% sucrose (in PBS, pH 7.2) for 10 min at room temperature, rinsed with PBS, and processed for TREK2 immunocytochemistry with the rabbit anti-TREK2 antibody from Alomone Labs as described above.

    Techniques: In Vivo, Injection, Expressing, Western Blot, Fluorescence, Staining, Binding Assay, Immunostaining

    A , SF rate was related to Em in C-neurons in neuropathic pain models. SF rate in intact L4 C-nociceptors was significantly linearly correlated with Em 7 d after SNA and mSNA surgery (see Materials and Methods and Results). B , C , SFL 1 d after CFA was related to ipsilateral TREK2 levels. In ipsilateral L5 DRG small neurons in 5 rats 1 d after CFA-induced cutaneous inflammation, there were significant linear correlations of ( B ) SFLd and ( C ) number of SFLn with median ipsilateral L5 small DRG neuron TREK2 pixel densities for each rat, whether in entire cytoplasm, or edge, mid, or inner cytoplasmic regions. Thus, SFL after CFA in individual rats was related to ipsilateral TREK2 levels in small DRG neurons.

    Journal: The Journal of Neuroscience

    Article Title: TREK2 Expressed Selectively in IB4-Binding C-Fiber Nociceptors Hyperpolarizes Their Membrane Potentials and Limits Spontaneous Pain

    doi: 10.1523/JNEUROSCI.4528-13.2014

    Figure Lengend Snippet: A , SF rate was related to Em in C-neurons in neuropathic pain models. SF rate in intact L4 C-nociceptors was significantly linearly correlated with Em 7 d after SNA and mSNA surgery (see Materials and Methods and Results). B , C , SFL 1 d after CFA was related to ipsilateral TREK2 levels. In ipsilateral L5 DRG small neurons in 5 rats 1 d after CFA-induced cutaneous inflammation, there were significant linear correlations of ( B ) SFLd and ( C ) number of SFLn with median ipsilateral L5 small DRG neuron TREK2 pixel densities for each rat, whether in entire cytoplasm, or edge, mid, or inner cytoplasmic regions. Thus, SFL after CFA in individual rats was related to ipsilateral TREK2 levels in small DRG neurons.

    Article Snippet: Three days after transfection, the HEK cell cultures expressing the different K2P channels were washed with PBS, fixed with 4% paraformaldehyde in 4.2% sucrose (in PBS, pH 7.2) for 10 min at room temperature, rinsed with PBS, and processed for TREK2 immunocytochemistry with the rabbit anti-TREK2 antibody from Alomone Labs as described above.

    Techniques:

    TREK/TRAAK channel expression in DRG neurons and transfected cells. ( A ) Western blot on DRG lysates isolated from wild-type (WT) or TREK1, TREK2, TRAAK triple KO (3KO) mice. Channel proteins were detected by using anti-TREK1, anti-TREK2, or anti-TRAAK antibodies. Bands consistent with the calculated molecular mass were detected at 45 kDa (TREK1), 60 kDa (TREK2), and 43 kDa (TRAAK) from wild-type DRG neurons but not from neurons isolated from TREK1/TREK2/TRAAK triple KO mice. In Lower , detection of β-tubulin. ( B ) Immunodetection of TREK1 (green) and TREK2 (red) in rat DRG neurons. Colocalization produces yellow in the merge image. (Scale bar: 50 µm.) ( C ) Coimmunoprecipitation following HA-TREK1 and Myc-TREK2 or TRAAK cotransfection in MDCK cells. HA-TREK1 is detected in Myc-TREK2 as well in TRAAK precipitates. Inputs represent 2% of the total cell lysate.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Mixing and matching TREK/TRAAK subunits generate heterodimeric K2P channels with unique properties

    doi: 10.1073/pnas.1522748113

    Figure Lengend Snippet: TREK/TRAAK channel expression in DRG neurons and transfected cells. ( A ) Western blot on DRG lysates isolated from wild-type (WT) or TREK1, TREK2, TRAAK triple KO (3KO) mice. Channel proteins were detected by using anti-TREK1, anti-TREK2, or anti-TRAAK antibodies. Bands consistent with the calculated molecular mass were detected at 45 kDa (TREK1), 60 kDa (TREK2), and 43 kDa (TRAAK) from wild-type DRG neurons but not from neurons isolated from TREK1/TREK2/TRAAK triple KO mice. In Lower , detection of β-tubulin. ( B ) Immunodetection of TREK1 (green) and TREK2 (red) in rat DRG neurons. Colocalization produces yellow in the merge image. (Scale bar: 50 µm.) ( C ) Coimmunoprecipitation following HA-TREK1 and Myc-TREK2 or TRAAK cotransfection in MDCK cells. HA-TREK1 is detected in Myc-TREK2 as well in TRAAK precipitates. Inputs represent 2% of the total cell lysate.

    Article Snippet: The neurons were then incubated overnight with anti-TREK1 and anti-TREK2 antibodies (1/200; Alomone Labs) followed with incubation with FITC-conjugated goat anti-rabbit IgG (1/500) and Cy-3–conjugated rabbit anti-goat IgG (1/250).

    Techniques: Expressing, Transfection, Western Blot, Isolation, Mouse Assay, Immunodetection, Cotransfection

    RR sensitivity of various TREK1, TREK2, and Td-TREK1/TREK2 in Xenopus oocytes. Current–voltage relationship is shown for each condition recorded under control bath solution perfusion (ND96, black circle), potassium symmetric condition (80 mM K + , gray circle), and under perfusion of 10 µM RR in 80 mM K + (white triangle). TREK1, Td-TREK1/TREK1, TREK2 D135I (containing the corresponding residue of the RR-resistant TREK1), Td-TREK1/TREK2, Td-TREK2/TREK1, Td-TREK1/TREK2 D135I , and Td-TREK2 D135I /TREK1 are insensitive to RR as shown by the overlapping between the IV curves in 80 mM K + with or without RR. TREK2, Td-TREK2/TREK2, TREK1 I110D (containing the corresponding residue of the RR-sensitive TREK2), Td-TREK1 I110D /TREK2, and Td-TREK2/TREK1 I110D are sensitive to RR as illustrated by the decrease in current amplitude with 10 μM RR compared with 80 mM K + bath solution.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Mixing and matching TREK/TRAAK subunits generate heterodimeric K2P channels with unique properties

    doi: 10.1073/pnas.1522748113

    Figure Lengend Snippet: RR sensitivity of various TREK1, TREK2, and Td-TREK1/TREK2 in Xenopus oocytes. Current–voltage relationship is shown for each condition recorded under control bath solution perfusion (ND96, black circle), potassium symmetric condition (80 mM K + , gray circle), and under perfusion of 10 µM RR in 80 mM K + (white triangle). TREK1, Td-TREK1/TREK1, TREK2 D135I (containing the corresponding residue of the RR-resistant TREK1), Td-TREK1/TREK2, Td-TREK2/TREK1, Td-TREK1/TREK2 D135I , and Td-TREK2 D135I /TREK1 are insensitive to RR as shown by the overlapping between the IV curves in 80 mM K + with or without RR. TREK2, Td-TREK2/TREK2, TREK1 I110D (containing the corresponding residue of the RR-sensitive TREK2), Td-TREK1 I110D /TREK2, and Td-TREK2/TREK1 I110D are sensitive to RR as illustrated by the decrease in current amplitude with 10 μM RR compared with 80 mM K + bath solution.

    Article Snippet: The neurons were then incubated overnight with anti-TREK1 and anti-TREK2 antibodies (1/200; Alomone Labs) followed with incubation with FITC-conjugated goat anti-rabbit IgG (1/500) and Cy-3–conjugated rabbit anti-goat IgG (1/250).

    Techniques:

    Dominant-negative TRAAK and TREK1 subunits coexpressed with K 2P channels in Xenopus oocytes. ( A ) TRAAK DN , containing a loss-of-function mutation in the pore region, G106E, has a dominant negative effect on TREK/TRAAK currents but not on THIK1. Representative current traces obtained from oocytes expressing TRAAK, TREK1, TREK2, and THIK1 (2.5 ng cRNA per oocyte) alone or in with TRAAK DN (7.5 ng). Currents were recorded at membrane potentials ranging from –120 mV to +60 mV from a holding potential of –80 mV in 10-mV increments. ( B ) TREK1 DN , containing a loss-of-function mutation in the pore region, G144E, has a dominant negative effect on TREK/TRAAK currents but not on THIK1, TWIK1, or TASK3. Wild-type TREK1, TREK2, TRAAK, THIK1, and TASK3 and an active mutant of TWIK1 locked at the cell surface (TWIK1-K274E-I293A,I294A) were coinjected in a 1:3 ratio with TREK1 DN in oocytes or expressed alone for comparison. Steady-state average current amplitudes at 0 mV were normalized with current amplitude means obtained when the same K 2P subunits was expressed alone. Conditions with dominant negatives were compared with the relative K 2P subunits expressed alone and analyzed by using a two-sample t test: * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Mixing and matching TREK/TRAAK subunits generate heterodimeric K2P channels with unique properties

    doi: 10.1073/pnas.1522748113

    Figure Lengend Snippet: Dominant-negative TRAAK and TREK1 subunits coexpressed with K 2P channels in Xenopus oocytes. ( A ) TRAAK DN , containing a loss-of-function mutation in the pore region, G106E, has a dominant negative effect on TREK/TRAAK currents but not on THIK1. Representative current traces obtained from oocytes expressing TRAAK, TREK1, TREK2, and THIK1 (2.5 ng cRNA per oocyte) alone or in with TRAAK DN (7.5 ng). Currents were recorded at membrane potentials ranging from –120 mV to +60 mV from a holding potential of –80 mV in 10-mV increments. ( B ) TREK1 DN , containing a loss-of-function mutation in the pore region, G144E, has a dominant negative effect on TREK/TRAAK currents but not on THIK1, TWIK1, or TASK3. Wild-type TREK1, TREK2, TRAAK, THIK1, and TASK3 and an active mutant of TWIK1 locked at the cell surface (TWIK1-K274E-I293A,I294A) were coinjected in a 1:3 ratio with TREK1 DN in oocytes or expressed alone for comparison. Steady-state average current amplitudes at 0 mV were normalized with current amplitude means obtained when the same K 2P subunits was expressed alone. Conditions with dominant negatives were compared with the relative K 2P subunits expressed alone and analyzed by using a two-sample t test: * P

    Article Snippet: The neurons were then incubated overnight with anti-TREK1 and anti-TREK2 antibodies (1/200; Alomone Labs) followed with incubation with FITC-conjugated goat anti-rabbit IgG (1/500) and Cy-3–conjugated rabbit anti-goat IgG (1/250).

    Techniques: Dominant Negative Mutation, Mutagenesis, Expressing

    TREK1, TREK2, and TRAAK interact in transfected MDCK cells. ( A ) Immunolocalization of HA-TREK1a (green) and Myc-TREK2c (red) or TRAAK (red). ( B ) Coimmunoprecipitation experiments. Inputs represent 2% of the total cell lysate. ( C ) FRET experiments. Different combinations of TREK1, TREK2, TRAAK, and THIK1 fused to enhanced cyan fluorescent protein (eCFP) and enhanced yellow fluorescent protein (eYFP) were coexpressed. eCFP directly linked to eYFP in pTOM serves as positive control. FRET was measured at the plasma membrane. Number of cells is given in parentheses. Data were analyzed by using one-way ANOVA with post hoc multiple comparisons using the Tukey’s test: * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Mixing and matching TREK/TRAAK subunits generate heterodimeric K2P channels with unique properties

    doi: 10.1073/pnas.1522748113

    Figure Lengend Snippet: TREK1, TREK2, and TRAAK interact in transfected MDCK cells. ( A ) Immunolocalization of HA-TREK1a (green) and Myc-TREK2c (red) or TRAAK (red). ( B ) Coimmunoprecipitation experiments. Inputs represent 2% of the total cell lysate. ( C ) FRET experiments. Different combinations of TREK1, TREK2, TRAAK, and THIK1 fused to enhanced cyan fluorescent protein (eCFP) and enhanced yellow fluorescent protein (eYFP) were coexpressed. eCFP directly linked to eYFP in pTOM serves as positive control. FRET was measured at the plasma membrane. Number of cells is given in parentheses. Data were analyzed by using one-way ANOVA with post hoc multiple comparisons using the Tukey’s test: * P

    Article Snippet: The neurons were then incubated overnight with anti-TREK1 and anti-TREK2 antibodies (1/200; Alomone Labs) followed with incubation with FITC-conjugated goat anti-rabbit IgG (1/500) and Cy-3–conjugated rabbit anti-goat IgG (1/250).

    Techniques: Transfection, Positive Control

    Contribution of each TREK/TRAAK monomer to the formation of heterodimers. ( A ) Dominant-negative TRAAK mutant bearing a loss of function mutation (TRAAK DN , TRAAK G106E) in the first pore domain was coexpressed with TRAAK, TREK1, TREK2, and THIK1 in oocytes (3:1 ratio, 7.5 ng/2.5 ng) and current recorded at membrane potentials ranging from –120 mV to +60 mV from a holding potential of – 80 mV in 10-mV increments. Histograms represent normalized current at 0 mV for oocytes expressing K 2P subunit alone (gray) or with TRAAK DN (black). Two-sample t test: *** P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Mixing and matching TREK/TRAAK subunits generate heterodimeric K2P channels with unique properties

    doi: 10.1073/pnas.1522748113

    Figure Lengend Snippet: Contribution of each TREK/TRAAK monomer to the formation of heterodimers. ( A ) Dominant-negative TRAAK mutant bearing a loss of function mutation (TRAAK DN , TRAAK G106E) in the first pore domain was coexpressed with TRAAK, TREK1, TREK2, and THIK1 in oocytes (3:1 ratio, 7.5 ng/2.5 ng) and current recorded at membrane potentials ranging from –120 mV to +60 mV from a holding potential of – 80 mV in 10-mV increments. Histograms represent normalized current at 0 mV for oocytes expressing K 2P subunit alone (gray) or with TRAAK DN (black). Two-sample t test: *** P

    Article Snippet: The neurons were then incubated overnight with anti-TREK1 and anti-TREK2 antibodies (1/200; Alomone Labs) followed with incubation with FITC-conjugated goat anti-rabbit IgG (1/500) and Cy-3–conjugated rabbit anti-goat IgG (1/250).

    Techniques: Dominant Negative Mutation, Mutagenesis, Expressing

    Single-channel properties of TREK/TRAAK homomeric and heteromeric channels in transfected HEK293 cells. ( A ) Electrophysiological properties of TREK1, TREK2 and Td-TREK1/TREK2. ( Left ) Single-channel recordings at −60 mV. Currents were recorded in cell-attached patches held at pipette potentials from +100 mV to −100 mV in bath solution containing 150 mM KCl. ( Middle ) Single-channel current–voltage relationships ( n = 10). ( Right ) Unitary conductances at −100 and +100 mV ( n = 10). ( B ) Electrophysiological properties of TREK1, TRAAK, and Td-TREK1/TRAAK. ( Left ) Single-channel recordings from cells expressing TREK1, TRAAK, and Td-TREK1/TRAAK at −60 mV. Currents were recorded as in A . ( Middle ) Single-channel current–voltage relationships of the different channels ( n = 10). ( Right ) Unitary conductances at -100 and +100 mV ( n = 10). ( C ) Current–voltage relationships of mean currents. The mean currents ( I = NPoi) were obtained from cell-attached macropatches ( n = 5). Mann–Whitney test: ** P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Mixing and matching TREK/TRAAK subunits generate heterodimeric K2P channels with unique properties

    doi: 10.1073/pnas.1522748113

    Figure Lengend Snippet: Single-channel properties of TREK/TRAAK homomeric and heteromeric channels in transfected HEK293 cells. ( A ) Electrophysiological properties of TREK1, TREK2 and Td-TREK1/TREK2. ( Left ) Single-channel recordings at −60 mV. Currents were recorded in cell-attached patches held at pipette potentials from +100 mV to −100 mV in bath solution containing 150 mM KCl. ( Middle ) Single-channel current–voltage relationships ( n = 10). ( Right ) Unitary conductances at −100 and +100 mV ( n = 10). ( B ) Electrophysiological properties of TREK1, TRAAK, and Td-TREK1/TRAAK. ( Left ) Single-channel recordings from cells expressing TREK1, TRAAK, and Td-TREK1/TRAAK at −60 mV. Currents were recorded as in A . ( Middle ) Single-channel current–voltage relationships of the different channels ( n = 10). ( Right ) Unitary conductances at -100 and +100 mV ( n = 10). ( C ) Current–voltage relationships of mean currents. The mean currents ( I = NPoi) were obtained from cell-attached macropatches ( n = 5). Mann–Whitney test: ** P

    Article Snippet: The neurons were then incubated overnight with anti-TREK1 and anti-TREK2 antibodies (1/200; Alomone Labs) followed with incubation with FITC-conjugated goat anti-rabbit IgG (1/500) and Cy-3–conjugated rabbit anti-goat IgG (1/250).

    Techniques: Transfection, Transferring, Expressing, MANN-WHITNEY

    All TREK1 and TREK2 isoforms assemble with similar efficiency in MDCK cells. ( A ) Representative images obtained by microscopy for TREK1 isoforms produced by AS of exon 1, TREK1a, b, c, and d, or by ATI, TREK1ΔNt, coexpressed with TREK2c isoform and used for PLA measurements. Green cells corresponding to cells expressing TREK1 or transfected with empty pIRES2-eGFP vector were counted positives when costained with red PLA probes. ( B ) TREK2 variants generated by AS (TREK2a, b, and c) or ATI (TREK2M60, M72, starting at methionine 60 or 72 numbered from TREK2c) were coexpressed with TREK1b. Representative microscopic images obtained for PLA and used for quantification.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Mixing and matching TREK/TRAAK subunits generate heterodimeric K2P channels with unique properties

    doi: 10.1073/pnas.1522748113

    Figure Lengend Snippet: All TREK1 and TREK2 isoforms assemble with similar efficiency in MDCK cells. ( A ) Representative images obtained by microscopy for TREK1 isoforms produced by AS of exon 1, TREK1a, b, c, and d, or by ATI, TREK1ΔNt, coexpressed with TREK2c isoform and used for PLA measurements. Green cells corresponding to cells expressing TREK1 or transfected with empty pIRES2-eGFP vector were counted positives when costained with red PLA probes. ( B ) TREK2 variants generated by AS (TREK2a, b, and c) or ATI (TREK2M60, M72, starting at methionine 60 or 72 numbered from TREK2c) were coexpressed with TREK1b. Representative microscopic images obtained for PLA and used for quantification.

    Article Snippet: The neurons were then incubated overnight with anti-TREK1 and anti-TREK2 antibodies (1/200; Alomone Labs) followed with incubation with FITC-conjugated goat anti-rabbit IgG (1/500) and Cy-3–conjugated rabbit anti-goat IgG (1/250).

    Techniques: Microscopy, Produced, Proximity Ligation Assay, Expressing, Transfection, Plasmid Preparation, Generated

    TREK1 and TREK2 isoforms assemble in MDCK cells. ( A ) TREK1 isoforms produced by AS of exon 1, TREK1a, b, c, and d, or by ATI, TREK1ΔNt, were coexpressed with TREK2c isoform and used for PLA measurements. Quantitative measurement is illustrated by a bar graph with the partial N-ter aa sequences of the various mouse TREK1 isoforms. MKWK sequence is the beginning of the first membrane-spanning domain M1. The gap in the N-ter corresponds to 40 residues. ( B ) TREK2 variants generated by AS (TREK2a, b, and c) or ATI (TREK2M60, M72, starting at methionine 60 or 72 numbered from TREK2c) were coexpressed with TREK1b. PLA quantification is given with the N-ter sequences of the various mouse TREK2 isoforms. The gap in the N-ter corresponds to 36 residues. Three independent experiments were performed that correspond to > 250 cells analyzed per condition.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Mixing and matching TREK/TRAAK subunits generate heterodimeric K2P channels with unique properties

    doi: 10.1073/pnas.1522748113

    Figure Lengend Snippet: TREK1 and TREK2 isoforms assemble in MDCK cells. ( A ) TREK1 isoforms produced by AS of exon 1, TREK1a, b, c, and d, or by ATI, TREK1ΔNt, were coexpressed with TREK2c isoform and used for PLA measurements. Quantitative measurement is illustrated by a bar graph with the partial N-ter aa sequences of the various mouse TREK1 isoforms. MKWK sequence is the beginning of the first membrane-spanning domain M1. The gap in the N-ter corresponds to 40 residues. ( B ) TREK2 variants generated by AS (TREK2a, b, and c) or ATI (TREK2M60, M72, starting at methionine 60 or 72 numbered from TREK2c) were coexpressed with TREK1b. PLA quantification is given with the N-ter sequences of the various mouse TREK2 isoforms. The gap in the N-ter corresponds to 36 residues. Three independent experiments were performed that correspond to > 250 cells analyzed per condition.

    Article Snippet: The neurons were then incubated overnight with anti-TREK1 and anti-TREK2 antibodies (1/200; Alomone Labs) followed with incubation with FITC-conjugated goat anti-rabbit IgG (1/500) and Cy-3–conjugated rabbit anti-goat IgG (1/250).

    Techniques: Produced, Proximity Ligation Assay, Sequencing, Generated