polyclonal primary tert antibody  (Bioss)

Journal: Scientific Reports

Article Title: Trpc1 as the Missing Link Between the Bmp and Ca 2+ Signalling Pathways During Neural Specification in Amphibians

doi: 10.1038/s41598-019-52556-0

Figure Lengend Snippet: trpc1 is expressed in the ectoderm of blastula and gastrula stage embryos. Spatial expression of trpc1 mRNA in blastula and gastrula stage embryos. Whole mount in situ hybridization was performed on embryos fixed at blastula (stage 9), early gastrula (stage 10.5) and late gastrula (stage 11.5). (A) Expression of trpc1 at stage 9 in an intact embryo (animal pole view), showing that the ectoderm is labelled. (B) Sagittal section taken through the embryo shown in panel A, indicating trpc1 expression in the ectoderm alone; the mesoderm and endoderm were not labelled. (C) Expression of trpc1 in an intact embryo at stage 10.5 (animal pole view). (D) Sagittal section taken through the embryo shown in panel (C) , showing trpc1 expression in the ectoderm, with no difference in the level of expression between the dorsal and the ventral sides. (E) Expression of trpc1 in an intact embryo at stage 11.5 (animal pole view). (F) Sagittal section taken through the embryo shown in panel E. AP, VP, ect, mes, end and Bl are animal pole, vegetal pole, ectoderm, mesoderm endoderm and blastocoel, respectively. In panels (D) and (F) , dorsal is to the right and the arrows indicate the blastopore lip. Scale bar is 300 µm.

Article Snippet: Trpc1 localization was revealed with the rabbit anti-Trpc1 polyclonal primary antibody (NB100–91315, Novus Biologicals; at a 1/20 dilution), and an Alexa-488-conjugated anti-rabbit secondary antibody (A11008, Thermo Fisher Scientific).

Techniques: Expressing, In Situ Hybridization

Journal: Scientific Reports

Article Title: Trpc1 as the Missing Link Between the Bmp and Ca 2+ Signalling Pathways During Neural Specification in Amphibians

doi: 10.1038/s41598-019-52556-0

Figure Lengend Snippet: Pattern of localization of Trpc1 in gastrula stage embryos. (A) Single optical view of a sagittal section taken though a gastrula stage embryo, showing the localization of Trpc1 in the ectoderm (ect) and mesoderm (mes). There is a lower level of Trpc1 expression in the endoderm (end). The arrow indicates the position of the blastopore lip. The regions bounded by the dashed white rectangles are shown at higher magnification in panels (B–D). These images show that at the cellular level, Trpc1 is localized mainly in the plasma membrane. Trpc1 localization, shown in green, was revealed with the rabbit anti-Trpc1 polyclonal antibody and an Alexa-555-conjugated anti-rabbit secondary antibody. The nuclei (in blue) were labelled with ToPro3. Scale bars are 300 µm in panel (A), and 40 µm in panels (B–D).

Article Snippet: Trpc1 localization was revealed with the rabbit anti-Trpc1 polyclonal primary antibody (NB100–91315, Novus Biologicals; at a 1/20 dilution), and an Alexa-488-conjugated anti-rabbit secondary antibody (A11008, Thermo Fisher Scientific).

Techniques: Expressing, Clinical Proteomics, Membrane

Journal: Scientific Reports

Article Title: Trpc1 as the Missing Link Between the Bmp and Ca 2+ Signalling Pathways During Neural Specification in Amphibians

doi: 10.1038/s41598-019-52556-0

Figure Lengend Snippet: Trpc1 knock down reduces the expression of Trpc1 in the ectoderm and abolishes the increase in intracellular Ca 2+ generated following activation of Ca V 1.2 channels in animal cap explants. (A–D) Embryos at the 2-cell stage were injected with (A–C) control-MO or (B–D) TRPC1-MO1 into both blastomeres, and the expression of Trpc1 was revealed by immunostaining at the blastula stage. (A,B) Confocal view of sagittal section taken through the entire embryo. ( C,D ) Confocal view at the level of the ectoderm. trpc1 knock-down impaired the expression of Trpc1. The nuclei (in blue) were labelled with ToPro3. Scale bars are 350 µm in (A,B) and 40 µm in (C,D) . (E,F) Relative changes in fluorescence (F/F0) revealing changes in intracellular Ca 2+ generated in single animal caps loaded with the Ca 2+ -indicator Fluo4 and isolated from embryos injected with either (E) control-MO or (F) TRPC1-MO1. The data are plotted as the mean of F/F0 (red traces) + SEM (black bars) from 7 (E) or 10 (F) randomly selected fields within a single animal cap. Noggin (2 µg/mL) was added (blue arrows) within the first 10 min after the start of data acquisition. Additional data are provided in Figure .

Article Snippet: Trpc1 localization was revealed with the rabbit anti-Trpc1 polyclonal primary antibody (NB100–91315, Novus Biologicals; at a 1/20 dilution), and an Alexa-488-conjugated anti-rabbit secondary antibody (A11008, Thermo Fisher Scientific).

Techniques: Knockdown, Expressing, Generated, Activation Assay, Injection, Control, Immunostaining, Fluorescence, Isolation

Journal: Scientific Reports

Article Title: Trpc1 as the Missing Link Between the Bmp and Ca 2+ Signalling Pathways During Neural Specification in Amphibians

doi: 10.1038/s41598-019-52556-0

Figure Lengend Snippet: Membrane depolarisation induced by noggin in the ectoderm requires Trpc1. Control morpholino (CMO) or TRPC1-MO1 was injected into both blastomeres of 2-cell stage embryos. Animal caps were then prepared at late blastula (stage 9) and loaded with the potentiometric dye DiBAC4(3). (A,B) The mean calculated membrane potential revealed membrane depolarisation following the addition of noggin (3 µg/mL, see blue arrow) in animal caps prepared from (A) CMO- or (B) TRPC1-MO1-injected embryos (n = 4 for each). (C,D) The mean calculated membrane potential in isolated ectoderm cells (n = 5 cells) that were dissociated from animal caps prepared from (C) CMO- or (D) TRPC1-MO1-injected embryos. (E) A box plot showing the maximal depolarisation values reached after noggin stimulation of animal caps or dissociated ectoderm cells prepared from CMO-injected embryos (white bars) or TRPC1-MO1-injected embryos (grey bars). The maximal depolarisation values for the animal caps and dissociated cells prepared from CMO- and TRPC1-MO1 embryos were significantly different, (Mann-Whitney test with *P < 0.02 for animal cap recordings, and **P < 0.004 for dissociated cell recordings).

Article Snippet: Trpc1 localization was revealed with the rabbit anti-Trpc1 polyclonal primary antibody (NB100–91315, Novus Biologicals; at a 1/20 dilution), and an Alexa-488-conjugated anti-rabbit secondary antibody (A11008, Thermo Fisher Scientific).

Techniques: Membrane, Control, Injection, Isolation, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Trpc1 as the Missing Link Between the Bmp and Ca 2+ Signalling Pathways During Neural Specification in Amphibians

doi: 10.1038/s41598-019-52556-0

Figure Lengend Snippet: trpc1 knock-down impairs the expression of the early neural gene, zic3 . Embryos were co-injected at the 8-cell stage into a single dorsal animal blastomere with nuclear β-galactosidase mRNA and: (A) the standard control-MO, (B) a splice-blocking MO (TRPC1 -MO3), (C) TRPC1 - MO1, or (D) TRPC1- MO1 plus r-trpc1 mRNA. Embryos were then fixed at stage 14 for subsequent whole-mount in situ hybridization of zic3; see red arrowheads in panel (A) . The side of the embryo injected with MO ± r-trpc1- mRNA was confirmed by reaction of β-galactosidase with X-Gal, as shown by the blue labelling on the right side of each embryo; see white arrowhead in panel (A) . Scale bar is 500 µm. (E) A bar chart showing the mean ± S.E.M. (n = 17) ratio of the area of zic3 expression on the injected and un-injected sides of the embryo. The asterisks indicate data that are significantly different at p < 0.001 when using one-way ANOVA and the Tukey’s honest significance post-hoc test.

Article Snippet: Trpc1 localization was revealed with the rabbit anti-Trpc1 polyclonal primary antibody (NB100–91315, Novus Biologicals; at a 1/20 dilution), and an Alexa-488-conjugated anti-rabbit secondary antibody (A11008, Thermo Fisher Scientific).

Techniques: Knockdown, Expressing, Injection, Control, Blocking Assay, In Situ Hybridization

Journal: Scientific Reports

Article Title: Trpc1 as the Missing Link Between the Bmp and Ca 2+ Signalling Pathways During Neural Specification in Amphibians

doi: 10.1038/s41598-019-52556-0

Figure Lengend Snippet: The tail domain of BMPRII is essential for the interaction with Trpc1, and noggin modulates the BMPRII-Trpc1 interaction. (A) Schematic illustration of the full-length BMPRII and the tail domain-deleted construct (BRII-∆TD). The yellow, black, grey and red rectangles show the signal peptide (SP); transmembrane domain (TM), kinase domain, and tail domain respectively. (B) Representative western blots showing the immunoprecipitation data acquired when analysing the protein-protein interaction between BMPRII and Trpc1. Embryos at the 4-cell stage were either injected (+) or not (−) with BMPRII-HA (200 pg/cell) or BRII-∆TD-HA (200 pg/cell), along with Myc-Trpc1 (200 pg/cell) into all the blastomeres. Animal caps were then prepared at stage 8–9, lysed and subjected to immunoprecipitation. ns, non-specific band. Western blot data were revealed by enhanced chemiluminescence (ECL; n = 4 independent experiments). (C ) Representative western blots showing the interaction between BMPRII and TRPC1 in the presence of noggin. Animal caps were collected at stage 8–9 from embryos injected with BMPRII-HA (200 pg/cell) and Myc-Trpc1 (200 pg/cell) into all the blastomeres at the 4-cell stage, after which they were either incubated (+) or not (−) with 2 µg/mL noggin for 15 min, and then lysed and subjected to immunoprecipitation with anti-Myc antibody. The western blot data were revealed by ECL (n = 3 independent experiments). (D) Quantification of the BMPRII-HA fraction associated with Myc-Trpc1. Ratios of co-immunoprecipitated BMPRII-HA: Myc-Trpc1 were calculated in the absence or presence of noggin using the Bio-Rad ChemiDoc Image Lab software 5.2.1. There data represent the mean ± from 3 independent experiments. The asterisk indicates data that are significantly different at p < 0.05 when using the Mann-Whitney test. The full-length blots are presented in Supplementary figure .

Article Snippet: Trpc1 localization was revealed with the rabbit anti-Trpc1 polyclonal primary antibody (NB100–91315, Novus Biologicals; at a 1/20 dilution), and an Alexa-488-conjugated anti-rabbit secondary antibody (A11008, Thermo Fisher Scientific).

Techniques: Construct, Western Blot, Immunoprecipitation, Injection, Incubation, Software, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Trpc1 as the Missing Link Between the Bmp and Ca 2+ Signalling Pathways During Neural Specification in Amphibians

doi: 10.1038/s41598-019-52556-0

Figure Lengend Snippet: Hypothetical model to depict the role of Trpc1 channels in linking the inhibition of BMP pathway by noggin, and the activation of Ca v 1.2 channels in ectodermal cells. During gastrulation, the cells of the embryonic ectoderm have the choice between two fates; they can give rise to either epidermal or neural progenitors. In the plasma membrane, the molecular components involved in this choice are BMP receptors type I (BmprI) and type II (BmprII), Trpc1 and voltage-dependent Ca 2+ channels (Ca v 1.2). The membrane potential in the ectoderm is ~−60 mV; i.e., the interior is negatively charged . ( A ) Induction of the epidermis occurs through a signalling cascade, which involves the binding of Bmp4 to its receptor, and then the transphosphorylation of BmprII by BmprI. This is followed by the activation of Smads, which translocate into the nucleus to form active transcriptional complexes to control the expression of epidermal genes. In this scenario, there is no interaction between Trpc1 and BmprII, and Ca v 1.2 remains inactive. ( B ) During neural induction, noggin binds to BMP4, and thus prevents the activation of the BMP pathway. As a consequence, it induces a physical interaction between BmprII and Trpc1 channels. This interaction leads to the activation of Trpc1, which either alone or associated with other Trp channels (e.g., Trpv4), triggers an influx of cations (Ca 2+ and Na + ). This influx of cations depolarizes the membrane (i.e., there is more positive charge inside) up to a threshold sufficient to open the voltage-gated Ca 2+ channel, Ca v 1.2. As we have previously shown , , the resulting influx of Ca 2+ is then sufficient to activate the expression of downstream neural specific genes, such as prmt1b , zic3 and sox2 .

Article Snippet: Trpc1 localization was revealed with the rabbit anti-Trpc1 polyclonal primary antibody (NB100–91315, Novus Biologicals; at a 1/20 dilution), and an Alexa-488-conjugated anti-rabbit secondary antibody (A11008, Thermo Fisher Scientific).

Techniques: Inhibition, Activation Assay, Clinical Proteomics, Membrane, Binding Assay, Control, Expressing