Journal: PLoS ONE

Article Title: Rat Hypocretin/Orexin Neurons Are Maintained in a Depolarized State by TRPC Channels

doi: 10.1371/journal.pone.0015673

Figure Lengend Snippet: (A–C) In voltage-clamp configuration, intracellular application of either a TRPC1 (A) or a TRPC3 (B) or a TRPC4 (C) antibody (AbTRPC1, AbTRPC3 and AbTRPC4 respectively, all from Alomone Labs) has no effect on the current needed to clamp the cell at −50 mV. (D–E) Intracellular application of a TRPC5 antibody (Alomone Labs), in contrast, induces an outward current (D) that is blocked by co-application of a TRPC5 antigen (E). (F–G) In current-clamp configuration, intracellular application of a TRPC5 antibody alone (from either Alomone Labs (F) or NeuroMab (inset in F) induces an important membrane hyperpolarization and cessation of firing. In contrast, intracellular co-application of the TRPC5 antibody (Alomone Labs) together with its appropriate antigen (AgTRPC5) has no effect (G, to compare with F). (H) Voltage-clamp ramps in absence (Ctrl) and presence of a TRPC5 antibody (AbTRPC5, Alomone Labs). In the inset, subtraction of voltage-clamp ramps (Ctrl - AbTRPC5) suggests the presence of a voltage-dependent TRPC5 current.

Article Snippet: References and validation (based on physiology, western blotting, immunohistochemistry, antisense and siRNA approaches) for the Alomone antibodies used in the present study (i.e. for TRPC1, 3, 4 and 5) can be found in the following papers: for TRPC1 – ; for TRPC3 , , , , ; for TRPC4 – , ; for TRPC5 , , – .

Techniques:

Journal: PLoS ONE

Article Title: Rat Hypocretin/Orexin Neurons Are Maintained in a Depolarized State by TRPC Channels

doi: 10.1371/journal.pone.0015673

Figure Lengend Snippet: (A) Transient (2 min) bath-application of 100 µM flufenamic acid (FFA), a non specific blocker of cationic currents, produces a strong membrane hyperpolarization and cessation of firing; inset: in voltage-clamp this hyperpolarization corresponds to an outward current. (B) Voltage-clamp ramps in absence (Ctrl) and presence (FFA) of flufenamic acid. The subtraction of voltage-clamp ramps shown in the inset suggests the presence of a voltage-dependent cationic current. (C–D) In hcrt/orx neurons loaded with a TRPC5 antibody, bath-application of FFA had only a small additional effect either in current-clamp (C) or in voltage-clamp (D).

Article Snippet: References and validation (based on physiology, western blotting, immunohistochemistry, antisense and siRNA approaches) for the Alomone antibodies used in the present study (i.e. for TRPC1, 3, 4 and 5) can be found in the following papers: for TRPC1 – ; for TRPC3 , , , , ; for TRPC4 – , ; for TRPC5 , , – .

Techniques:

Journal: PLoS ONE

Article Title: Rat Hypocretin/Orexin Neurons Are Maintained in a Depolarized State by TRPC Channels

doi: 10.1371/journal.pone.0015673

Figure Lengend Snippet: (A–B) Photomicrographs from a dual-immunostained section combining an hcrt/orx antibody from Santa Cruz (A) and a TRPC5 antibody from Alomone Labs (B). Examples of double labelled hcrt/orx-TRPC5 cells are indicated by white arrowheads and one such cell by a double circle (enlarged in the corresponding small panels to the right). Some hcrt/orx cells do not express TRPC5, as shown by an example indicated with a single circle. It is noteworthy that many hcrt/orx-negative cells also expressed TRPC5, as shown by an example indicated with a downward arrow (in panels A and B). (C–D) Same as in A and B with an hcrt/orx antibody from Phoenix Pharmaceuticals (C) and a TRPC5 antibody from NeuroMab (D). (Scale bars: 20 µm).

Article Snippet: References and validation (based on physiology, western blotting, immunohistochemistry, antisense and siRNA approaches) for the Alomone antibodies used in the present study (i.e. for TRPC1, 3, 4 and 5) can be found in the following papers: for TRPC1 – ; for TRPC3 , , , , ; for TRPC4 – , ; for TRPC5 , , – .

Techniques:

Journal: PLoS ONE

Article Title: Rat Hypocretin/Orexin Neurons Are Maintained in a Depolarized State by TRPC Channels

doi: 10.1371/journal.pone.0015673

Figure Lengend Snippet: (A–C) In voltage-clamp configuration, intracellular application of either a TRPC1 (A) or a TRPC3 (B) or a TRPC4 (C) antibody (AbTRPC1, AbTRPC3 and AbTRPC4 respectively, all from Alomone Labs) has no effect on the current needed to clamp the cell at −50 mV. (D–E) Intracellular application of a TRPC5 antibody (Alomone Labs), in contrast, induces an outward current (D) that is blocked by co-application of a TRPC5 antigen (E). (F–G) In current-clamp configuration, intracellular application of a TRPC5 antibody alone (from either Alomone Labs (F) or NeuroMab (inset in F) induces an important membrane hyperpolarization and cessation of firing. In contrast, intracellular co-application of the TRPC5 antibody (Alomone Labs) together with its appropriate antigen (AgTRPC5) has no effect (G, to compare with F). (H) Voltage-clamp ramps in absence (Ctrl) and presence of a TRPC5 antibody (AbTRPC5, Alomone Labs). In the inset, subtraction of voltage-clamp ramps (Ctrl - AbTRPC5) suggests the presence of a voltage-dependent TRPC5 current.

Article Snippet: The hyperpolarizing action of the TRPC5 antibody (from Alomone Labs) was checked with an antibody from a different source (NeuroMab, used in also applied intracellularly at 6 ng/µl which yielded similar results (inset of , mean ΔV ± SEM = −12.08±1.56 mV, n = 4; Student's t test Alomone versus NeuroMab p = 0.62).

Techniques:

Journal: PLoS ONE

Article Title: Rat Hypocretin/Orexin Neurons Are Maintained in a Depolarized State by TRPC Channels

doi: 10.1371/journal.pone.0015673

Figure Lengend Snippet: (A) Transient (2 min) bath-application of 100 µM flufenamic acid (FFA), a non specific blocker of cationic currents, produces a strong membrane hyperpolarization and cessation of firing; inset: in voltage-clamp this hyperpolarization corresponds to an outward current. (B) Voltage-clamp ramps in absence (Ctrl) and presence (FFA) of flufenamic acid. The subtraction of voltage-clamp ramps shown in the inset suggests the presence of a voltage-dependent cationic current. (C–D) In hcrt/orx neurons loaded with a TRPC5 antibody, bath-application of FFA had only a small additional effect either in current-clamp (C) or in voltage-clamp (D).

Article Snippet: The hyperpolarizing action of the TRPC5 antibody (from Alomone Labs) was checked with an antibody from a different source (NeuroMab, used in also applied intracellularly at 6 ng/µl which yielded similar results (inset of , mean ΔV ± SEM = −12.08±1.56 mV, n = 4; Student's t test Alomone versus NeuroMab p = 0.62).

Techniques:

Journal: PLoS ONE

Article Title: Rat Hypocretin/Orexin Neurons Are Maintained in a Depolarized State by TRPC Channels

doi: 10.1371/journal.pone.0015673

Figure Lengend Snippet: (A–B) Photomicrographs from a dual-immunostained section combining an hcrt/orx antibody from Santa Cruz (A) and a TRPC5 antibody from Alomone Labs (B). Examples of double labelled hcrt/orx-TRPC5 cells are indicated by white arrowheads and one such cell by a double circle (enlarged in the corresponding small panels to the right). Some hcrt/orx cells do not express TRPC5, as shown by an example indicated with a single circle. It is noteworthy that many hcrt/orx-negative cells also expressed TRPC5, as shown by an example indicated with a downward arrow (in panels A and B). (C–D) Same as in A and B with an hcrt/orx antibody from Phoenix Pharmaceuticals (C) and a TRPC5 antibody from NeuroMab (D). (Scale bars: 20 µm).

Article Snippet: The hyperpolarizing action of the TRPC5 antibody (from Alomone Labs) was checked with an antibody from a different source (NeuroMab, used in also applied intracellularly at 6 ng/µl which yielded similar results (inset of , mean ΔV ± SEM = −12.08±1.56 mV, n = 4; Student's t test Alomone versus NeuroMab p = 0.62).

Techniques:

Journal: PLoS ONE

Article Title: Rat Hypocretin/Orexin Neurons Are Maintained in a Depolarized State by TRPC Channels

doi: 10.1371/journal.pone.0015673

Figure Lengend Snippet: (A–C) In voltage-clamp configuration, intracellular application of either a TRPC1 (A) or a TRPC3 (B) or a TRPC4 (C) antibody (AbTRPC1, AbTRPC3 and AbTRPC4 respectively, all from Alomone Labs) has no effect on the current needed to clamp the cell at −50 mV. (D–E) Intracellular application of a TRPC5 antibody (Alomone Labs), in contrast, induces an outward current (D) that is blocked by co-application of a TRPC5 antigen (E). (F–G) In current-clamp configuration, intracellular application of a TRPC5 antibody alone (from either Alomone Labs (F) or NeuroMab (inset in F) induces an important membrane hyperpolarization and cessation of firing. In contrast, intracellular co-application of the TRPC5 antibody (Alomone Labs) together with its appropriate antigen (AgTRPC5) has no effect (G, to compare with F). (H) Voltage-clamp ramps in absence (Ctrl) and presence of a TRPC5 antibody (AbTRPC5, Alomone Labs). In the inset, subtraction of voltage-clamp ramps (Ctrl - AbTRPC5) suggests the presence of a voltage-dependent TRPC5 current.

Article Snippet: Other sections were incubated with the rabbit anti-TRPC5 antibody (Alomone Labs) that had been preincubated with the corresponding control antigen (from Alomone Labs, in a ratio of 1∶3 for 2 hours at room temperature). (The control antigen for the TRPC5 antibody from NeuroMab was not available.)

Techniques:

Journal: PLoS ONE

Article Title: Rat Hypocretin/Orexin Neurons Are Maintained in a Depolarized State by TRPC Channels

doi: 10.1371/journal.pone.0015673

Figure Lengend Snippet: (A) Transient (2 min) bath-application of 100 µM flufenamic acid (FFA), a non specific blocker of cationic currents, produces a strong membrane hyperpolarization and cessation of firing; inset: in voltage-clamp this hyperpolarization corresponds to an outward current. (B) Voltage-clamp ramps in absence (Ctrl) and presence (FFA) of flufenamic acid. The subtraction of voltage-clamp ramps shown in the inset suggests the presence of a voltage-dependent cationic current. (C–D) In hcrt/orx neurons loaded with a TRPC5 antibody, bath-application of FFA had only a small additional effect either in current-clamp (C) or in voltage-clamp (D).

Article Snippet: Other sections were incubated with the rabbit anti-TRPC5 antibody (Alomone Labs) that had been preincubated with the corresponding control antigen (from Alomone Labs, in a ratio of 1∶3 for 2 hours at room temperature). (The control antigen for the TRPC5 antibody from NeuroMab was not available.)

Techniques:

Journal: PLoS ONE

Article Title: Rat Hypocretin/Orexin Neurons Are Maintained in a Depolarized State by TRPC Channels

doi: 10.1371/journal.pone.0015673

Figure Lengend Snippet: (A–B) Photomicrographs from a dual-immunostained section combining an hcrt/orx antibody from Santa Cruz (A) and a TRPC5 antibody from Alomone Labs (B). Examples of double labelled hcrt/orx-TRPC5 cells are indicated by white arrowheads and one such cell by a double circle (enlarged in the corresponding small panels to the right). Some hcrt/orx cells do not express TRPC5, as shown by an example indicated with a single circle. It is noteworthy that many hcrt/orx-negative cells also expressed TRPC5, as shown by an example indicated with a downward arrow (in panels A and B). (C–D) Same as in A and B with an hcrt/orx antibody from Phoenix Pharmaceuticals (C) and a TRPC5 antibody from NeuroMab (D). (Scale bars: 20 µm).

Article Snippet: Other sections were incubated with the rabbit anti-TRPC5 antibody (Alomone Labs) that had been preincubated with the corresponding control antigen (from Alomone Labs, in a ratio of 1∶3 for 2 hours at room temperature). (The control antigen for the TRPC5 antibody from NeuroMab was not available.)

Techniques:

Journal: eLife

Article Title: Unfolding and identification of membrane proteins in situ

doi: 10.7554/eLife.77427

Figure Lengend Snippet: ( A ) Four clusters found in the dataset of force-distance (F-D) traces pulled from NG108-15 cells wild type. ( B ) The Bayesian identification of the clusters in ( A ) with the candidate membrane proteins. ( C ) Confocal images of NG108-15 cells overexpressing the construct N2b-Protein-GFP, as reported by the green fluorescence emitted by GFP. ( D ) From left to right, color density plots (blue indicating a colocalization among F-D traces of more than 80%) of the clusters superimposed to an F-D curve bearing the N2B signature (85 nm segment with a flat force below 10 pN) obtained in the sample overexpressing the candidate protein TMEM16F, TRPC1, TRPC5, and TRPC6, respectively. ( E ) Global histogram of L c of the clusters in A and the clusters with the N2B signature from cells transfected with the corresponding construct. ( F ) Position of the most likely rupture regions of the proteins according to ( E ) superimposed to the cartoon of the cryo electron microscopy (cryo-EM) structure available in the PDB.

Article Snippet: Antibody , Anti-TRPC5 (Rabbit polyclonal) , Alomone Labs , Cat#ACC-020 , WB(1:500).

Techniques: Construct, Fluorescence, Transfection, Electron Microscopy, Cryo-EM Sample Prep

Journal: eLife

Article Title: Unfolding and identification of membrane proteins in situ

doi: 10.7554/eLife.77427

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-TRPC5 (Rabbit polyclonal) , Alomone Labs , Cat#ACC-020 , WB(1:500).

Techniques: Plasmid Preparation, Modification, Transfection, Construct, Sequencing, Labeling, Isolation, Recombinant, Software, Staining

Journal: eLife

Article Title: Unfolding and identification of membrane proteins in situ

doi: 10.7554/eLife.77427

Figure Lengend Snippet: ( A ) Four clusters found in the dataset of force-distance (F-D) traces pulled from NG108-15 cells wild type. ( B ) The Bayesian identification of the clusters in ( A ) with the candidate membrane proteins. ( C ) Confocal images of NG108-15 cells overexpressing the construct N2b-Protein-GFP, as reported by the green fluorescence emitted by GFP. ( D ) From left to right, color density plots (blue indicating a colocalization among F-D traces of more than 80%) of the clusters superimposed to an F-D curve bearing the N2B signature (85 nm segment with a flat force below 10 pN) obtained in the sample overexpressing the candidate protein TMEM16F, TRPC1, TRPC5, and TRPC6, respectively. ( E ) Global histogram of L c of the clusters in A and the clusters with the N2B signature from cells transfected with the corresponding construct. ( F ) Position of the most likely rupture regions of the proteins according to ( E ) superimposed to the cartoon of the cryo electron microscopy (cryo-EM) structure available in the PDB.

Article Snippet: The membranes were incubated with the corresponding primary antibodies 1:5000 for α-Tubulin (Cat# T8203, Sigma); 1:500 for TRPC1 (Cat# ACC-101, Alomone Labs)/5 (Cat# ACC-020, Alomone Labs)/6 (Cat#ACC-120, Alomone Labs) and 1:200 for TMEM16F (Cat# ACL-016, Alomone Labs) at 4°C overnight with gentle shaking.

Techniques: Construct, Fluorescence, Transfection, Electron Microscopy, Cryo-EM Sample Prep

Journal: eLife

Article Title: Unfolding and identification of membrane proteins in situ

doi: 10.7554/eLife.77427

Figure Lengend Snippet:

Article Snippet: The membranes were incubated with the corresponding primary antibodies 1:5000 for α-Tubulin (Cat# T8203, Sigma); 1:500 for TRPC1 (Cat# ACC-101, Alomone Labs)/5 (Cat# ACC-020, Alomone Labs)/6 (Cat#ACC-120, Alomone Labs) and 1:200 for TMEM16F (Cat# ACL-016, Alomone Labs) at 4°C overnight with gentle shaking.

Techniques: Plasmid Preparation, Modification, Transfection, Construct, Sequencing, Labeling, Isolation, Recombinant, Software, Staining