anti trek2 antibodies  (Alomone Labs)

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

Article Title: Mixing and matching TREK/TRAAK subunits generate heterodimeric K 2P 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, Immunodetection, Cotransfection

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

Article Title: Mixing and matching TREK/TRAAK subunits generate heterodimeric K 2P 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 < 0.05, **P < 0.01, and ***P < 0.001. (D) Duolink in situ PLA. Percentage of cells corresponds to the number of PLA-positive cells relative to the number of transfected cells. Number of independent experiments is given in parentheses and corresponds to >300 cells analyzed. Mann–Whitney test versus TREK1+CD4 as negative control: *P < 0.05.

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, In Situ, MANN-WHITNEY, Negative Control

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

Article Title: Mixing and matching TREK/TRAAK subunits generate heterodimeric K 2P 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, Sequencing, Generated

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

Article Title: Mixing and matching TREK/TRAAK subunits generate heterodimeric K 2P 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, Expressing, Transfection, Plasmid Preparation, Generated

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

Article Title: Mixing and matching TREK/TRAAK subunits generate heterodimeric K 2P 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 (TRAAKDN, 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 K2P subunit alone (gray) or with TRAAKDN (black). Two-sample t test: ***P < 0.001. (B) RR sensitivity of TREK1, TREK2, and covalent TREK1/TREK2 tandems (Td-TREK1/TREK2 and Td-TREK2/TREK1). I110D and D135I substitution correspond to mutation of the RR binding site in mouse TREK1a or TREK2c. Each cRNAs were injected alone in oocytes; currents were elicited as in A. For normalized current values, current measured at −100 mV in 80 mM K+ solution after addition of 10 µM RR was compared with current obtained in 80 mM K+ solution (control) and analyzed with paired Wilcoxon test: *P < 0.05. Number of oocytes is given in parentheses.

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, Binding Assay, Injection

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

Article Title: Mixing and matching TREK/TRAAK subunits generate heterodimeric K 2P channels with unique properties

doi: 10.1073/pnas.1522748113

Figure Lengend Snippet: Dominant-negative TRAAK and TREK1 subunits coexpressed with K2P channels in Xenopus oocytes. (A) TRAAKDN, 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 TRAAKDN (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) TREK1DN, 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 TREK1DN 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 K2P subunits was expressed alone. Conditions with dominant negatives were compared with the relative K2P subunits expressed alone and analyzed by using a two-sample t test: *P < 0.05 and ***P < 0.001. Number of oocytes is given in parentheses.

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

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

Article Title: Mixing and matching TREK/TRAAK subunits generate heterodimeric K 2P 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, TREK2D135I (containing the corresponding residue of the RR-resistant TREK1), Td-TREK1/TREK2, Td-TREK2/TREK1, Td-TREK1/TREK2D135I, and Td-TREK2D135I/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, TREK1I110D (containing the corresponding residue of the RR-sensitive TREK2), Td-TREK1I110D/TREK2, and Td-TREK2/TREK1I110D 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:

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

Article Title: Mixing and matching TREK/TRAAK subunits generate heterodimeric K 2P 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 < 0.01 and ***P < 0.001.

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