Journal: Journal of Cellular and Molecular Medicine

Article Title: Increased rhythmicity in hypertensive arterial smooth muscle is linked to transient receptor potential canonical channels

doi: 10.1111/j.1582-4934.2009.00890.x

Figure Lengend Snippet: Expression of TRPC1, TRPC3 and TRPC5 channels in mesenteric arterioles from SHR. Representative immunoblottings (A) and summary data (B) of TRPC1, TRPC3, TRPC4, TRPC5,TRPC6 channels and TRPC3 antibody with its respective antigenic peptide the protein expression in mesenteric arterioles from WKY (open bars) and SHR (filled bars). Data are mean ± S.E.M. of n = 4 independent experiments. * P < 0.05 or ** P < 0.01 by two-tailed Student’s t-test. (C) Light microscopy of mesenteric arterioles from SHR (upper panels) and immunohistochemistry (lower panels) of TRPC1, TRPC3 and TRPC5 protein expression in mesenteric arterioles from SHR. (D) Light microscopy of mesenteric arterioles from WKY (upper panels) and immunohistochemistry (lower panels) of TRPC1, TRPC3 and TRPC5 protein expression in mesenteric arterioles from WKY. Immunohistochemistry using specific antibodies labelled with green shows TRPC1, TRPC3 and TRPC5 protein expression in mesenteric arterioles. Control indicates immunohistochemistry after pre-incubation of the primary TRPC3 antibody with antigenic peptide for 12 hrs at 4°C. Bar denotes 200 μm. (43.0 ± 15%; P = n.s.; n = 6, ). In the presence of anti-TRPC5 antibodies the norepinephrine-induced vasomotion was not significantly affected (45.0 ± 11%; P = n.s. ). In the presence of anti-TRPC1 plus anti-TRPC3 antibodies the norepinephrine-induced vasomotion was significantly reduced to 29.0 ± 8% ( P < 0.05; n = 6, ). Furthermore, pre-incubation of mesenteric arterioles with anti-β-actin antibodies did not significantly affect norepinephrine-induced vasomotion when compared to control conditions (59.8 ± 15%, P = n.s.; n = 6, ). Incorporation of TRPC antibodies into mesenteric arteries by pinocytosis was facilitated by changing the bathing solution to a hypotonic solution according to recent literature .

Article Snippet: The membranes were incubated with primary rabbit antibodies against TRPC1, TRPC3, TRPC4, TRPC5 and TRPC6 (1:200; Alomone Labs), followed by incubation with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody.

Techniques: Expressing, Two Tailed Test, Light Microscopy, Immunohistochemistry, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Interactions between the Polysialylated Neural Cell Adhesion Molecule and the Transient Receptor Potential Canonical Channels 1, 4, and 5 Induce Entry of Ca 2+ into Neurons

doi: 10.3390/ijms231710027

Figure Lengend Snippet: NCAM and TRPC1, −4, and −5 interact via their ICDs. ( a , b ) A brain detergent extract was used for immunoprecipitation (IP) with TRPC1 antibody H-105, TRPC4/5 antibody H-80 (4/5), TRPC3/6/7 antibody H-100 (3/6/7), and nonimmune control antibody (Ig). The brain detergent extract (input) and the immunoprecipitates were subjected to Western blot (WB) analysis with NCAM antibody 5B8 (NCAM140/180) ( a ), NCAM antibody D3 (NCAM180), or TRPC3/6/7 antibody H-100 (3/6/7) ( b ). ( c ) Detergent extracts of membrane-enriched brain fractions (input) and TRPC1 antibody ACC-010 (1), TRPC4 antibody ACC-018 (4), TRPC5 antibody ACC-020 (5), and a nonimmune control antibody (Ig) were used for immunoprecipitation, and the NCAM antibody GTX133217 against the C-terminus (NCAM-CT), a chicken antibody against the extracellular domains of NCAM (NCAM-ECD), and PSA antibody 735 (PSA) were used for Western blot analysis. ( a – c ) Arrowheads indicate bands representing NCAM140, NCAM180, and polysialylated NCAM140 and NCAM180 (PSA-NCAM), and the arrow indicates TRPC3, −6, or −7 bands. ( d ) Immunoprecipitation was carried out with brain detergent extracts (input) from wild-type (+/+) and NCAM-deficient (−/−) mice using NCAM antibody H28 and followed by Western blot analysis with TRPC1 antibody E-6 (1), TRPC4 antibody N77/15 (4), TRPC5 antibody N67/15 (5), and TRPC3/6/7 antibody H-100 (3/6/7). Arrows indicate TRPC1, −4, or −5 bands. ( e , f ) NCAM antibody 5B8 (NCAM140/180), D3 (NCAM180) ( e ), and GTX133217 (NCAM-CT) ( f ) were used for Western blot analysis of precipitates from pull-down experiments using detergent brain extracts (input) ( e ) or a membrane-enriched fraction (input) ( f ) and beads only (ctrl) or beads with GST, GST-tagged N-terminal (1N), or C-terminal (1C) ICDs of TRPC1 or GST-tagged N-terminal ICDs of TRPC4 (4N) or TRPC5 (5N). Arrowheads indicate NCAM180 and PSA-NCAM bands. ( g – i ) TRPC1 antibody E-6 ( g , h ) or TRPC3/6/7 antibody H-100 ( i ) were used for Western blot analysis of precipitates from pull-down experiments with detergent brain extracts and His-tagged ICDs of NCAM140 (140) ( g – i ), NCAM180 (180) ( g , i ), L1 ( h , i ), or NCAM140 with deletion of the N-terminal (ΔN), middle (ΔM), or C-terminal (ΔC) part ( h ). Arrows indicate TRPC1 bands.

Article Snippet: The polyclonal rabbit anti-TRPC1 antibody ACC-010 against amino acids 557–571 in the intracellular loop of human TRPC1, rabbit anti-TRPC4 antibody ACC-018 against amino acids 943–958 in the C-terminal ICD of mouse TRPC1, the polyclonal rabbit anti-TRPC4 antibody ACC-119 against amino acids 458–469 in the second extracellular domain of rat TRPC4, and the polyclonal rabbit anti-TRPC5 antibody ACC-020 against amino acids 959–973 in the C-terminal ICD of human TRPC5 were from Alomone Labs (Jerusalem, Israel).

Techniques: Immunoprecipitation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Interactions between the Polysialylated Neural Cell Adhesion Molecule and the Transient Receptor Potential Canonical Channels 1, 4, and 5 Induce Entry of Ca 2+ into Neurons

doi: 10.3390/ijms231710027

Figure Lengend Snippet: NCAM140-ICD and NCAM180-ICD directly bind to the N-terminal ICDs of TRPC1, −4, and −5. Recombinant N-terminal ICDs of TRPC1 (TRPC1-ICD NT ) ( a , d ), TRPC4 (TRPC4-ICD NT ) ( b , e ), and TRPC5 (TRPC5-ICD NT ) ( c , f ) were substrate-coated and incubated with increasing concentrations of recombinant NCAM140-ICD ( a – c ), NCAM180-ICD ( a – c ), CHL1-ICD ( a – c ), or NCAM140-ICD with a deletion of the N-terminal (NCAM140ΔN), middle (NCAM140ΔM), or C-terminal (NCAM140ΔC) parts ( d – f ). Binding was determined by ELISA using NCAM antibody P61 ( a – c ), CHL1 antibody ( a – c ), and NCAM antibody GTX133217 ( d – f ). Mean values ± SEM from 3 independent experiments carried out in triplicates are shown.

Article Snippet: The polyclonal rabbit anti-TRPC1 antibody ACC-010 against amino acids 557–571 in the intracellular loop of human TRPC1, rabbit anti-TRPC4 antibody ACC-018 against amino acids 943–958 in the C-terminal ICD of mouse TRPC1, the polyclonal rabbit anti-TRPC4 antibody ACC-119 against amino acids 458–469 in the second extracellular domain of rat TRPC4, and the polyclonal rabbit anti-TRPC5 antibody ACC-020 against amino acids 959–973 in the C-terminal ICD of human TRPC5 were from Alomone Labs (Jerusalem, Israel).

Techniques: Recombinant, Incubation, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Interactions between the Polysialylated Neural Cell Adhesion Molecule and the Transient Receptor Potential Canonical Channels 1, 4, and 5 Induce Entry of Ca 2+ into Neurons

doi: 10.3390/ijms231710027

Figure Lengend Snippet: Colominic acid/PSA binds to the N-terminal ICDs of TRPC1, −4, and −5, and its binding site overlaps with the binding site of NCAM140-ICD. Recombinant N-terminal ICDs of TRPC1 (TRPC1-ICD NT ) ( a , d ), TRPC4 (TRPC4-ICD NT ) ( b ), TRPC5 (TRPC5-ICD NT ) ( c ), and the TRPC1 peptide ( d ) were substrate-coated and incubated with increasing concentrations of colominic acid/PSA or chondroitin sulfate. Binding was determined by ELISA with antibodies against PSA or chondroitin sulfate. Mean values ± SEM from 3 independent experiments carried out in triplicates are shown. TRPC1 peptide ( e ) or N-terminal TRPC1-ICD (TRPC1-ICD NT ) ( f ) were substrate-coated and incubated with increasing concentrations of NCAM140-ICD or CHL1-ICD ( e ) or with one NCAM140-ICD concentration and different concentrations of TRPC1 peptide ( f ). Binding was determined by ELISA using NCAM antibody P61 ( e , f ) and CHL1 antibody C-18 ( e ). Mean values ± SEM from 3 independent experiments carried out in triplicates are shown.

Article Snippet: The polyclonal rabbit anti-TRPC1 antibody ACC-010 against amino acids 557–571 in the intracellular loop of human TRPC1, rabbit anti-TRPC4 antibody ACC-018 against amino acids 943–958 in the C-terminal ICD of mouse TRPC1, the polyclonal rabbit anti-TRPC4 antibody ACC-119 against amino acids 458–469 in the second extracellular domain of rat TRPC4, and the polyclonal rabbit anti-TRPC5 antibody ACC-020 against amino acids 959–973 in the C-terminal ICD of human TRPC5 were from Alomone Labs (Jerusalem, Israel).

Techniques: Binding Assay, Recombinant, Incubation, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Interactions between the Polysialylated Neural Cell Adhesion Molecule and the Transient Receptor Potential Canonical Channels 1, 4, and 5 Induce Entry of Ca 2+ into Neurons

doi: 10.3390/ijms231710027

Figure Lengend Snippet: NCAM colocalizes with TRPC1, −4, and −5 in hippocampal and cerebellar neurons. Cultured neurons were subjected to double immunostaining with rabbit NCAM antibody GTX133217 and mouse TRPC1 antibody E-6, TRPC4 antibody N77/15, or TRPC5 antibody N67/15, as well as with mouse PSA antibody 735 and rabbit TRPC1 antibody GTX54876 or rabbit TRPC4 antibody ACC-119. Nuclei were stained with DAPI (blue). ( a , b ) Representative images are shown for coimmunostaining of TRPC1 (red) and NCAM (green) or of TRPC1 (green) and PSA (red) in cerebellar ( a ) and hippocampal ( b ) neurons. Superimposition of red and green stainings shows colocalization in yellow. Scale bar: 20 µm. ( c , d ) Pearson’s coefficients were determined from 10 immunostained neurons per group and from 2 different cultures. Box plots show the Pearson’s coefficients and indicate an overlap of NCAM or PSA stainings with TRPC stainings in cerebellar ( c ) and hippocampal ( d ) neurons.

Article Snippet: The polyclonal rabbit anti-TRPC1 antibody ACC-010 against amino acids 557–571 in the intracellular loop of human TRPC1, rabbit anti-TRPC4 antibody ACC-018 against amino acids 943–958 in the C-terminal ICD of mouse TRPC1, the polyclonal rabbit anti-TRPC4 antibody ACC-119 against amino acids 458–469 in the second extracellular domain of rat TRPC4, and the polyclonal rabbit anti-TRPC5 antibody ACC-020 against amino acids 959–973 in the C-terminal ICD of human TRPC5 were from Alomone Labs (Jerusalem, Israel).

Techniques: Cell Culture, Double Immunostaining, Staining

Journal: International Journal of Molecular Sciences

Article Title: Interactions between the Polysialylated Neural Cell Adhesion Molecule and the Transient Receptor Potential Canonical Channels 1, 4, and 5 Induce Entry of Ca 2+ into Neurons

doi: 10.3390/ijms231710027

Figure Lengend Snippet: NCAM localizes in close proximity to TRPC1, −4, and −5 in hippocampal and cerebellar neurons. Cultured neurons from wild-type (NCAM+/+) and NCAM-deficient (NCAM−/−) mice were subjected to proximity ligation assay with rabbit NCAM antibody GTX133217 and mouse TRPC1 antibody E-6, TRPC4 antibody N77/15, or TRPC5 antibody N67/15, as well as with mouse PSA antibody 735 and rabbit TRPC1 antibody GTX54876 or TRPC4 antibody ACC-119. Nuclei were stained with DAPI (blue). Representative images are shown for TRPC1, −4, or −5 and NCAM or PSA in cerebellar ( a ) and hippocampal ( c ) neurons (scale bar: 20 µm). Red dots indicate close proximity of NCAM or PSA with TRPCs in cerebellar ( b ) and hippocampal ( d ) neurons. ( b , d ) Numbers of red dots were determined from 2 independent experiments analyzing 10 neurons per group and experiment. Scatter plots show mean values ± SEM and single values for the numbers of red dots per cell (*** p < 0.001 relative to wild-type neurons; Mann–Whitney test).

Article Snippet: The polyclonal rabbit anti-TRPC1 antibody ACC-010 against amino acids 557–571 in the intracellular loop of human TRPC1, rabbit anti-TRPC4 antibody ACC-018 against amino acids 943–958 in the C-terminal ICD of mouse TRPC1, the polyclonal rabbit anti-TRPC4 antibody ACC-119 against amino acids 458–469 in the second extracellular domain of rat TRPC4, and the polyclonal rabbit anti-TRPC5 antibody ACC-020 against amino acids 959–973 in the C-terminal ICD of human TRPC5 were from Alomone Labs (Jerusalem, Israel).

Techniques: Cell Culture, Proximity Ligation Assay, Staining, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Interactions between the Polysialylated Neural Cell Adhesion Molecule and the Transient Receptor Potential Canonical Channels 1, 4, and 5 Induce Entry of Ca 2+ into Neurons

doi: 10.3390/ijms231710027

Figure Lengend Snippet: NCAM localizes in close proximity to TRPC1, −4, and −5 in cortical neurons. ( a , b ) Cultured cortical neurons from wild-type (NCAM+/+) and NCAM-deficient (NCAM−/−) mice were subjected to proximity ligation assay with rabbit NCAM antibody GTX133217 and mouse TRPC1 antibody E-6, TRPC4 antibody N77/15, or TRPC5 antibody N67/15, as well as with mouse PSA antibody 735 and rabbit TRPC1 antibody GTX54876 or TRPC4 antibody ACC-119. Nuclei were stained with DAPI (blue). ( a ) Representative images are shown for proximity ligation of TRPCs and NCAM or PSA in wild-type neurons (scale bar: 20 µm). Red dots indicate close proximity of NCAM or PSA with TRPCs in wild-type, but not NCAM-deficient neurons. Numbers of red dots from 3 independent experiments analyzing 10 neurons per group and experiment were determined. ( b ) Scatter plots show mean values ± SEM and single values for numbers of red dots per cell (*** p < 0.001 relative to wild-type neurons; Mann–Whitney test).

Article Snippet: The polyclonal rabbit anti-TRPC1 antibody ACC-010 against amino acids 557–571 in the intracellular loop of human TRPC1, rabbit anti-TRPC4 antibody ACC-018 against amino acids 943–958 in the C-terminal ICD of mouse TRPC1, the polyclonal rabbit anti-TRPC4 antibody ACC-119 against amino acids 458–469 in the second extracellular domain of rat TRPC4, and the polyclonal rabbit anti-TRPC5 antibody ACC-020 against amino acids 959–973 in the C-terminal ICD of human TRPC5 were from Alomone Labs (Jerusalem, Israel).

Techniques: Cell Culture, Proximity Ligation Assay, Staining, Ligation, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Interactions between the Polysialylated Neural Cell Adhesion Molecule and the Transient Receptor Potential Canonical Channels 1, 4, and 5 Induce Entry of Ca 2+ into Neurons

doi: 10.3390/ijms231710027

Figure Lengend Snippet: NCAM colocalizes with TRPC1, −4, and −5 predominantly in the plasma membrane of cortical neurons. ( a ) The scheme illustrates the principle of TIRF microscopy. A glass coverslip (black bar) and an adhering neuron (light blue) with nucleus and ER (ovals) are depicted. Application of laser light (light green; red arrows) at a certain angle leads to total reflection of light at the interfaces of the glass coverslip and the aqueous film between coverslip and adherent cells, generating an evanescent wave, which creates an evanescent field (yellow area). As the energy in this field decreases exponentially with distance of typically 60–100 nm to the interfaces, only fluorophores in this proximity to the coverslip are excited, leaving other fluorophores in the cell not excited. This allows the detection of protein interactions at the plasma membrane and in the ER membranes near the plasma membrane. Shown within the evanescent field are excited fluorophores indicating TRPC/NCAM interactions (red circles), plasma membrane (PM) marker proteins (blue circles), and ER membrane marker proteins (green circles). Outside of this field, light grey, dark grey, and black circles indicate the corresponding nonexcited fluorophores. ( b – e ) Cultured wild-type cortical neurons were first transfected with plasmids encoding a plasma membrane-associated fusion protein of blue fluorescence protein and the CAAX motif-containing sequence of H-Ras or encoding an ER membrane-associated fusion protein of enhanced green fluorescence protein, the ER-targeting sequence of calreticulin, and the ER retention sequence KDEL. Subsequently, the cells were treated without ( d , e ) or with the NCAM antibody ( d ) or EndoN ( e ) and subjected to proximity ligation with rabbit NCAM antibody GTX133217 and mouse TRPC1 antibody E-6, TRPC4 antibody N77/15, or TRPC5 antibody N67/15 or with mouse PSA antibody 735 and rabbit TRPC1 antibody ACC-010 or TRPC4 antibody ACC-018. The neurons were then analyzed by TIRF microscopy. ( b ) Representative images show TRPC1/NCAM interactions (red dots) at the cell surface predominantly near the plasma membrane marker protein (blue) (NCAM/TRPC1/PM), but rarely near the ER membrane marker protein (green) (NCAM/TRPC1/ER) (scale bar: 20 µm). ( c ) Representative images are shown for interactions of TRPCs with NCAM or PSA at the cell surface (scale bar: 20 µm). The numbers of red dots near to the plasma membrane marker (blue) or to the ER marker (green) were counted separately (overlapping dots were not considered) and indicate the interaction of NCAM or PSA with TRPCs in the plasma membrane or in ER membranes near the plasma membrane. ( d , e ) Scatter plots show mean values ± SEM and single values for the numbers of plasma membrane-associated (PM) and ER-associated (ER) red dots per cell from 3 independent experiments analyzing 10 neurons per group and experiment (*** p < 0.001; Mann–Whitney test comparing PM and ER values).

Article Snippet: The polyclonal rabbit anti-TRPC1 antibody ACC-010 against amino acids 557–571 in the intracellular loop of human TRPC1, rabbit anti-TRPC4 antibody ACC-018 against amino acids 943–958 in the C-terminal ICD of mouse TRPC1, the polyclonal rabbit anti-TRPC4 antibody ACC-119 against amino acids 458–469 in the second extracellular domain of rat TRPC4, and the polyclonal rabbit anti-TRPC5 antibody ACC-020 against amino acids 959–973 in the C-terminal ICD of human TRPC5 were from Alomone Labs (Jerusalem, Israel).

Techniques: Microscopy, Marker, Cell Culture, Transfection, Fluorescence, Sequencing, Ligation, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Interactions between the Polysialylated Neural Cell Adhesion Molecule and the Transient Receptor Potential Canonical Channels 1, 4, and 5 Induce Entry of Ca 2+ into Neurons

doi: 10.3390/ijms231710027

Figure Lengend Snippet: NCAM regulates the Ca 2+ entry via TRPC1, −4, and −5 in transfected CHO cells that express PSA-NCAM. NCAM-expressing CHO 2A10 cells, PSA-NCAM-expressing CHO C6 cells, or NCAM-lacking NCAM neg CHO cells were mock-transfected or transfected with plasmids encoding TRPC1/4 or TRPC1/5 heteromers, loaded with the Ca 2+ indicator Fluo-4 AM, treated with or without function-triggering NCAM antibody in the absence or presence of the TRPC inhibitor SKF96365, and subjected to Ca 2+ imaging. ( a ) A representative time course of a Ca 2+ response of mock-transfected and TRPC1/4- or TRPC1/5-transfected CHO C6 cells after application of a function-triggering NCAM antibody (arrow; NCAM Ab) is shown (* p < 0.05, ** p < 0.01; mixed effects analysis comparing values from the TRPC1/4- or TRPC1/5-transfected cells with the mock-transfected cells). ( b – f ) Scatter plots show mean values ± SEM and single values for the peak Fluo-4 AM intensity values of 6–8 cells per treatment for mock-transfected and TRPC1/4- or TRPC1/5-transfected CHO C6 cells ( b , e ), CHO 2A10 cells ( c , f ), and NCAM-lacking NCAM neg CHO cells and in the absence ( b – f ) and presence ( e , f ) of SKF96365 (SKF) (* p < 0.05, ** p < 0.01 relative to mock-transfected ( b ) or vehicle-treated ( e ) cells; one-way ANOVA with Kruskal–Wallis post hoc test ( b – d ), Mann–Whitney test ( e ); ns, not significant). ( g – i ) Lysates of CHO 2A10 and CHO C6 cells were used for immunoprecipitation (IP) with TRPC1 antibody ACC-010 ( g ), TRPC4 antibody ACC-018 ( h ), TRPC5 antibody ACC-020 ( h ), and antibody GTX133217 against the C-terminus of NCAM (NCAM-CT) ( i ), and nonimmune control antibodies (Ig) ( g – i ) were used for immunoprecipitation. The lysates (input) and the immunoprecipitates were subjected to Western blot analysis with NCAM antibody GTX133217 (NCAM-CT) ( g , h ) or TRPC4 antibody ACC-018 ( i ).

Article Snippet: The polyclonal rabbit anti-TRPC1 antibody ACC-010 against amino acids 557–571 in the intracellular loop of human TRPC1, rabbit anti-TRPC4 antibody ACC-018 against amino acids 943–958 in the C-terminal ICD of mouse TRPC1, the polyclonal rabbit anti-TRPC4 antibody ACC-119 against amino acids 458–469 in the second extracellular domain of rat TRPC4, and the polyclonal rabbit anti-TRPC5 antibody ACC-020 against amino acids 959–973 in the C-terminal ICD of human TRPC5 were from Alomone Labs (Jerusalem, Israel).

Techniques: Transfection, Expressing, Imaging, MANN-WHITNEY, Immunoprecipitation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Interactions between the Polysialylated Neural Cell Adhesion Molecule and the Transient Receptor Potential Canonical Channels 1, 4, and 5 Induce Entry of Ca 2+ into Neurons

doi: 10.3390/ijms231710027

Figure Lengend Snippet: NCAM regulates the Ca 2+ entry in cortical neurons via TRPC1, −4, and −5. Cortical neurons from wild-type (NCAM+/+) and NCAM-deficient (NCAM−/−) mice were treated without or with the PSA-degrading enzyme EndoN, loaded with Fluo-4 AM, preincubated with colominic acid/PSA (CA) or the TRPC inhibitors Pico145, HC-070, M084, or GSK-417651A, treated with or without function-triggering NCAM antibody, and subjected to Ca 2+ imaging. Mean values ± SEM from 3 independent experiments analyzing 10 cells per treatment and experiment are shown for Ca 2+ responses after treatment with function-triggering NCAM antibody (arrow; NCAM Ab) of wild-type and NCAM-deficient neurons ( a ), of non-treated and EndoN-treated wild-type neurons ( b ), and of wild-type neurons treated with or without Pico145 ( c ). ( d ) Pico145 reduces the NCAM-stimulated Ca 2+ response in a concentration-dependent manner. Mean values ± SEM from 3 experiments analyzing 10 cells per experiment are shown. ( e ) Scatter plots show the peak Fluo-4 AM intensity values for wild-type neurons (WT), NCAM-deficient (KO) neurons, and EndoN-pretreated wild-type neurons (EndoN) after NCAM antibody treatment in the absence (vehicle) or presence of Pico145 (Pico), HC-070 (HC070), M084, GSK-417651A (GSK), or colominic acid/PSA (CA). Mean values ± SEM and single values from 3 experiments analyzing 10 cells per treatment and experiment are shown (** p < 0.01, *** p < 0.001, **** p < 0.0001 relative to values from vehicle-treated wild-type neurons; one-way ANOVA with Kruskal–Wallis post hoc test). ( f ) Representative time courses are shown for the Ca 2+ responses after application of wild-type neurons with cell-penetrating unmutated tat-TRPC1/WT and mutated tat-TRPC1/mut peptides (arrow; TRPC1 peptides). ( g ) Cortical neurons were pretreated with thapsigargin in the absence of Ca 2+ , loaded with Fluo-4 AM, treated with NCAM antibody in the presence of Ca 2+ , and analyzed by Ca 2+ imaging. Representative Ca 2+ responses of thapsigargin-treated neurons after treatment with NCAM antibody (arrow; NCAM Ab) in the absence (thapsigargin/no Ca 2+ ) or presence (thasigargin/Ca 2+ ) of Ca 2+ and of neurons not treated with thapsigargin and treated with NCAM antibody in the absence of Ca 2+ (no thasigargin/no Ca 2+ ) are shown. ( h ) Brain extracts from wild-type (+/+) and NCAM-deficient (−/−) mice were subjected to Western blot analysis with TRPC5 antibody 1C8, TRPC4 antibody ACC-018, TRPC1 antibody E-6, and α-tubulin antibody. TRPC1, −4, and −5 levels relative to α-tubulin levels were determined.

Article Snippet: The polyclonal rabbit anti-TRPC1 antibody ACC-010 against amino acids 557–571 in the intracellular loop of human TRPC1, rabbit anti-TRPC4 antibody ACC-018 against amino acids 943–958 in the C-terminal ICD of mouse TRPC1, the polyclonal rabbit anti-TRPC4 antibody ACC-119 against amino acids 458–469 in the second extracellular domain of rat TRPC4, and the polyclonal rabbit anti-TRPC5 antibody ACC-020 against amino acids 959–973 in the C-terminal ICD of human TRPC5 were from Alomone Labs (Jerusalem, Israel).

Techniques: Imaging, Concentration Assay, Western Blot

Journal: Cells

Article Title: Transient Receptor Potential Canonical 5-Scramblase Signaling Complex Mediates Neuronal Phosphatidylserine Externalization and Apoptosis

doi: 10.3390/cells9030547

Figure Lengend Snippet: Association between TRPC5 and PLSCR1 in freshly isolated mouse cortical neurons. ( A , B ) Representative images of co-immunoprecipitation experiments. ( A ): IP, anti-PLSCR1 antibody or IgG; IB, anti-TRPC5 antibody. B , IP, anti-TRPC5 antibody or IgG; IB, anti-PLSCR1 antibody. Immunoblots of cell lysates were also shown on the right. ( A ), immunoblot with rabbit anti-TRPC5 antibody; ( B ), immunoblot with goat anti-PLSCR1 antibody). The experiments were repeated for three times. ( C ) Representative images of co-immunoprecipitation experiments. Right: IP, anti-PLSCR1 antibody or IgG; IB, anti-TRPC1, anti-TRPC4, or anti-PLSCR1 antibody. Left, Immunoblots of cell lysates were also shown. ( D , E ) In situ proximity ligation assay (PLA) to identify the association between TRPC5 and PLSCR1 in native neurons. Representative images were in the presence of both anti-TRPC5 and anti-PLSCR1 antibodies ( E ), or in the presence of anti-TRPC5 antibody alone (Control) ( D ). In D and E , gray sign: anti-goat PLA probe Plus, green sign: goat anti-TRPC5 antibody, pink sign: anti-rabbit PLA probe Minus, blue sign: rabbit anti-PLSCR1 antibody, red dot: PLA signal, left oval: TRPC5 protein, right oval: PLSCR1 protein. Nuclei were stained blue by DAPI.

Article Snippet: Rabbit anti-TRPC5 (ACC-020), anti-TRPC4 (ACC-018) antibodies were purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Isolation, Immunoprecipitation, Western Blot, In Situ, Proximity Ligation Assay, Staining