Journal: PLoS ONE

Article Title: Increased Migration of Monocytes in Essential Hypertension Is Associated with Increased Transient Receptor Potential Channel Canonical Type 3 Channels

doi: 10.1371/journal.pone.0032628

Figure Lengend Snippet: A ; Immunoblot showing specificity of antibodies against TRPC3 in monocytes from normotensive control subjects (NT) and patients with essential hypertension (HT) in the absence or presence of TRPC3 antigens (TRPC3+Ag). The predicted molecular weight of TRPC3 is 97 kDa. B ; Immunoblot showing specificity of antibodies against TRPC3 in monocytes from normotensive control subjects (NT, n = 8), patients with type 2 diabetes mellitus (DM, n = 9), patients with essential hypertension (HT, n = 8) or hypertensive patients with type 2 diabetes mellitus (HT+DM, n = 10). Summary data of the TRPC3 expression (normalized to GAPDH). *p<0.05, compared to NT. Data are mean ± SEM. C ; Representative in-cell western assay and summary data of the TRPC3 expression (normalized to CD14 expression used as an internal reference) in monocytes from normotensive control subjects (Normotensive, and opened bars, n = 3) and patients with essential hypertension (Hypertensive, filled bars, n = 3) under control conditions and after transfection with scrambled siRNA or specific siRNA against TRPC3 for 48 h. In-cell western assay was performed using specific antibodies and fluorescence-labeled secondary antibodies. TRPC3 (visible in green) normalized to CD14 (used as an internal reference). Measurements were performed in duplicate for each sample. *p<0.05 or **p<0.01 for the comparison with their controls; and ## p<0.01 for the comparison Hypertensive (filled bars) vs. Normotensive (open bars). D ; Representative in-cell western assay and summary data of the TRPC3 and TRPC6 expression in monocytes from normotensive control subjects under control conditions and after transfection with specific siRNA against TRPC3 for 48 h. In-cell western assay was performed using specific antibodies and fluorescence-labeled secondary antibodies. TRPC3 and TRPC6 expression (visible in green) normalized to CD14 (visible in red used as an internal reference). Measurements were performed in duplicate for each sample. **p<0.01 compared to control conditions. Data are mean ± SEM of three independent experiments. E ; Summary data of the fMLP-induced monocyte migration from hypertensive patients (HT, filled bars) and normotensive control subjects (NT, opened bars) quantified by counting the number of cells that had completely migrated through the membrane in six random high-power fields (HPF, 40×) per well. Monocytes chemotaxis was expressed as the mean number of migrated cells per high-power fields from duplicate wells. Experiments were performed under control conditions, after transfection with scrambled siRNA or specific siRNA against TRPC3. *p<0.05; **p<0.01 compared to normotensive control subjects under control conditions. Data are mean ± SEM of eight independent experiments. F ; Spontaneous migrations of monocytes from normotensive control subjects (NT; open bars) and hypertensive patients (HT, filled bars) were tested using medium or after transfection with scrambled siRNA or specific siRNA against TRPC3. The data was quantified by counting the number of cells that had completely migrated through the membrane in six random high-power fields (HPF, 40×) per well. P>0.05 compared to NT. Data are percent of medium as mean ± SEM of three independent experiments.

Article Snippet: After that incubated with rabbit anti-TRPC3 antibodies (1∶1000, Alomone Laboratories, Jerusalem, Israel) as the primary antibodies for 2 h, washed, incubated with IRDye 800 CW infrared fluorescent dye conjugated goat anti-rabbit antibodies (1∶1000, Biomol, Hamburg, Germany) as the secondary antibody overnight and washed, and quantitative imaging was performed at 810 nm emission with an excitation wavelength of 780 nm.

Techniques: Western Blot, Molecular Weight, Expressing, In-Cell ELISA, Transfection, Fluorescence, Labeling, Migration, Chemotaxis Assay

Journal: PLoS ONE

Article Title: Increased Migration of Monocytes in Essential Hypertension Is Associated with Increased Transient Receptor Potential Channel Canonical Type 3 Channels

doi: 10.1371/journal.pone.0032628

Figure Lengend Snippet: A , B ; fMLP activates ERK or phosphorylation of ERK ( A ) and Akt or phosphorylation of Akt ( B ) in a dose- and time-dependent manner in monocytes from normotensive control subjects. 10 nmol/L open bars, 100 nmol/L filled bars. Data are mean ± SEM, n = 3. *p<0.05 compared to lower concentration conditions. C , D ; Increased fMLP-induced phosphorylation of ERK ( C ) and Akt ( D ) in monocytes from patients with essential hypertension. The proteins were measured using immunoblotting with specific antibodies. Data are mean ± SEM from three independent experiments. *p<0.05 compared to normotensive control subjects. E ; fMLP activates monocytes by an ERK-dependent and Akt-dependent pathway. Akt, ERK, or pERK and pAkt were measured using immunoblotting with specific antibodies. In the presence of 2-APB or after administration of specific siRNA against TRPC3, the fMLP-induced ERK, pERK; Akt and pAkt were significantly reduced when compared with control conditions. Data are mean ± SEM from six independent experiments. *p<0.05; **p<0.01 compared to control.

Article Snippet: After that incubated with rabbit anti-TRPC3 antibodies (1∶1000, Alomone Laboratories, Jerusalem, Israel) as the primary antibodies for 2 h, washed, incubated with IRDye 800 CW infrared fluorescent dye conjugated goat anti-rabbit antibodies (1∶1000, Biomol, Hamburg, Germany) as the secondary antibody overnight and washed, and quantitative imaging was performed at 810 nm emission with an excitation wavelength of 780 nm.

Techniques: Concentration Assay, Western Blot

Journal: Cell chemical biology

Article Title: Leveraging compound promiscuity to identify targetable cysteines within the kinome

doi: 10.1016/j.chembiol.2019.02.021

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The 10X running (Tris/Glycine/SDS buffer, 161–0772, Biorad), 10X transfer (35040, Thermo Fisher Scientific) and 20X TBS (sc-362305, Santa Cruz) buffers were diluted to 1X solutions prior to use.

Techniques: Recombinant, Plasmid Preparation, Transfection, Labeling, Expressing, Software, Protein Extraction, Bicinchoninic Acid Protein Assay, Peptide Fractionation, Staining

Journal:

Article Title: Neutralizing Antibodies Inhibit Axonal Spread of Herpes Simplex Virus Type 1 to Epidermal Cells In Vitro

doi:

Figure Lengend Snippet: Confocal micrographs of HSV-infected neurons stained for gC antigen at 15 hpi and after addition of human anti-gD monoclonal antibody (top) or control medium (bottom). The HSV inoculum (5 TCID50/cell) was aspirated after 1 h of incubation, and the cells were carefully washed once with HBSS. The HSV-infected or mock-infected dissociated neuronal cultures, incubated with a 1:2,500 dilution (400 ng/ml) of human anti-gD antibody, were fixed in 2.5% formaldehyde (ProSci Tech) in Sorensons buffer (pH 7.4) for 30 min and permeabilized with 0.1% Triton X-100 (Sigma) in PBS for 20 min. Nonspecific staining was blocked by incubation with 5% mouse serum in HBSS for 15 min. The cells on coverslips were then incubated with fluorescein isothiocyanate-conjugated anti-gC1 antibody (Syva Microtrak) (1:100 dilution), rinsed three times with HBSS, and mounted in mounting fluid (Syva Microtrak). Stained neurons were examined with a Bio-Rad MRC 600 confocal microscope. Note the similar distributions of gC antigen in the axon and cytoplasm in both micrographs. Bars, 40 μm (top) and 20 μm (bottom).

Article Snippet: The HSV-infected or mock-infected dissociated neuronal cultures, incubated with a 1:2,500 dilution (400 ng/ml) of human anti-gD antibody, were fixed in 2.5% formaldehyde (ProSci Tech) in Sorensons buffer (pH 7.4) for 30 min and permeabilized with 0.1% Triton X-100 (Sigma) in PBS for 20 min. Nonspecific staining was blocked by incubation with 5% mouse serum in HBSS for 15 min.

Techniques: Infection, Staining, Incubation, Microscopy

Journal:

Article Title: Differential Expression of Canonical (Classical) Transient Receptor Potential Channels in Guinea Pig Enteric Nervous System

doi: 10.1002/cne.21874

Figure Lengend Snippet: Primary Antibodies Used in the Study

Article Snippet: The sequence is H-RSESIAFIEESKKD ADEVFSS-NH2 Sheep 1:1,000 Chemicon/AB1529/0703053959 NPY Synthetic NPY peptide conjugated to bovine thyroglobulin Sheep 1:5,000 Chemicon/AB1583/0703053959 Synaptophysin Protein p38 6 from crude fractions of coated vesicles from bovine brain Mouse 1:200 Dako/M0776/00017473 TRPC1 Peptide QLYDK GYTSK EQKDC, corresponding to amino acid residues 557−571 of human TRPC1 (Accession {"type":"entrez-protein","attrs":{"text":"P48995","term_id":"1351302","term_text":"P48995"}} P48995 ) Rabbit 1:100 Alomone/ACC-010/AN-04 TRPC3 Peptide HKLSE KLNPS VLRC, corresponding to residues 822−835 of mouse TRPC3 (Accession {"type":"entrez-protein","attrs":{"text":"Q9QZC1","term_id":"10720320","term_text":"Q9QZC1"}} Q9QZC1 ) Rabbit 1:100 Alomone/ACC-016/AN-03 TRPC4 Peptide (C)KEKH AHEED SSIDY DL, corresponding to residues 943−958 of mouse TRPC4 (Accession {"type":"entrez-protein","attrs":{"text":"Q9QUQ5","term_id":"14548291","term_text":"Q9QUQ5"}} Q9QUQ5 ) Rabbit 1:100 Alomone/ACC-018/AN-01 TRPC5 Peptide (C)HKWGDGQEEQVTTRL corresponding to residues 959−973 of human TRPC5 (Accession {"type":"entrez-protein","attrs":{"text":"Q9UL62","term_id":"10720321","term_text":"Q9UL62"}} Q9UL62 ) Rabbit 1:100 Alomone/ACC-020/AN-07 TRPC6 Peptide (C)RRNE SQDYL LMDELG, corresponding to residues 24−38 of mouse TRPC6 (Accession {"type":"entrez-protein","attrs":{"text":"Q61143","term_id":"342187122","term_text":"Q61143"}} Q61143 ) Rabbit 1:100 Alomone/ACC-017/AN-02 TRPC7 Peptide ILKEL SKEEE DTDSS EEMLA, corresponding to residues 658−677 of human TRPC7 (Accession {"type":"entrez-nucleotide","attrs":{"text":"NM_020389","term_id":"262399373","term_text":"NM_020389"}} NM_020389 ) Rabbit 1:100 Chemicon/AB9326/VR1377347 VIP Synthetic peptide (1−28) coupled to KLH with glutaraldehyde Sheep 1:200 Chemicon/AB1581/0611044826 Open in a separate window Hu, anti-human neuronal protein; ChAT, choline-acetyltransferase; NeuN; neuronal nuclear protein; NOS; nitric oxide synthase; NPY, neuropeptide Y; TRPC, canonical transient receptor potential channel; VIP, vasoactive inhibitory peptide.

Techniques: Recombinant, Purification, Sequencing

Journal:

Article Title: Differential Expression of Canonical (Classical) Transient Receptor Potential Channels in Guinea Pig Enteric Nervous System

doi: 10.1002/cne.21874

Figure Lengend Snippet: TRPC protein expression in the guinea pig ENS. Western blot confirmed the expression of TRPC1 (120kDa), TRPC3 (∼95−100kDa), TRPC4 (∼95−100kDa), and TRPC6 (∼95−100kDa) proteins in LMMP and SMP of the guinea pig ileum, and TRPC1/4/6 proteins in dissociated myenteric ganglia. The immunoblots were at the same level as their corresponding positive controls (i.e., brain lysate). Immunoblots were either completely blocked or significantly reduced by preadsorbing the antibodies with their corresponding blocking peptides.

Article Snippet: The sequence is H-RSESIAFIEESKKD ADEVFSS-NH2 Sheep 1:1,000 Chemicon/AB1529/0703053959 NPY Synthetic NPY peptide conjugated to bovine thyroglobulin Sheep 1:5,000 Chemicon/AB1583/0703053959 Synaptophysin Protein p38 6 from crude fractions of coated vesicles from bovine brain Mouse 1:200 Dako/M0776/00017473 TRPC1 Peptide QLYDK GYTSK EQKDC, corresponding to amino acid residues 557−571 of human TRPC1 (Accession {"type":"entrez-protein","attrs":{"text":"P48995","term_id":"1351302","term_text":"P48995"}} P48995 ) Rabbit 1:100 Alomone/ACC-010/AN-04 TRPC3 Peptide HKLSE KLNPS VLRC, corresponding to residues 822−835 of mouse TRPC3 (Accession {"type":"entrez-protein","attrs":{"text":"Q9QZC1","term_id":"10720320","term_text":"Q9QZC1"}} Q9QZC1 ) Rabbit 1:100 Alomone/ACC-016/AN-03 TRPC4 Peptide (C)KEKH AHEED SSIDY DL, corresponding to residues 943−958 of mouse TRPC4 (Accession {"type":"entrez-protein","attrs":{"text":"Q9QUQ5","term_id":"14548291","term_text":"Q9QUQ5"}} Q9QUQ5 ) Rabbit 1:100 Alomone/ACC-018/AN-01 TRPC5 Peptide (C)HKWGDGQEEQVTTRL corresponding to residues 959−973 of human TRPC5 (Accession {"type":"entrez-protein","attrs":{"text":"Q9UL62","term_id":"10720321","term_text":"Q9UL62"}} Q9UL62 ) Rabbit 1:100 Alomone/ACC-020/AN-07 TRPC6 Peptide (C)RRNE SQDYL LMDELG, corresponding to residues 24−38 of mouse TRPC6 (Accession {"type":"entrez-protein","attrs":{"text":"Q61143","term_id":"342187122","term_text":"Q61143"}} Q61143 ) Rabbit 1:100 Alomone/ACC-017/AN-02 TRPC7 Peptide ILKEL SKEEE DTDSS EEMLA, corresponding to residues 658−677 of human TRPC7 (Accession {"type":"entrez-nucleotide","attrs":{"text":"NM_020389","term_id":"262399373","term_text":"NM_020389"}} NM_020389 ) Rabbit 1:100 Chemicon/AB9326/VR1377347 VIP Synthetic peptide (1−28) coupled to KLH with glutaraldehyde Sheep 1:200 Chemicon/AB1581/0611044826 Open in a separate window Hu, anti-human neuronal protein; ChAT, choline-acetyltransferase; NeuN; neuronal nuclear protein; NOS; nitric oxide synthase; NPY, neuropeptide Y; TRPC, canonical transient receptor potential channel; VIP, vasoactive inhibitory peptide.

Techniques: Expressing, Western Blot, Blocking Assay

Journal:

Article Title: Differential Expression of Canonical (Classical) Transient Receptor Potential Channels in Guinea Pig Enteric Nervous System

doi: 10.1002/cne.21874

Figure Lengend Snippet: Immunohistochemical demonstration of TRPC subunits in the ENS. TRPC subunits were labeled with indocarbocyanine (Cy3, red). The pan neuronal marker HuC/D (Hu) was labeled with fluorescein isothiocyanate (FITC, green). TRPC1 immunoreactivity (IR) was expressed in a subpopulation of neurons in the myenteric (A) and submucosal (B) plexuses. Double-labeling with an antibody for Hu confirmed that all TRPC1-IR cells are immunoreactive for Hu (A’, B’). TRPC3-IR was not found in the myenteric plexus (C, C’), but was expressed in a small neuronal population in submucosal ganglia (D) and was colocalized with Hu-IR (D’). TRPC4-IR was found in neuronal cell bodies and nerve fibers in both the myenteric (E) and submucosal (F) plexuses. TRPC4-immunoreactive nerve cell bodies were colocalized with Hu (E’, F’). TRPC6- IR was found in neuronal cell bodies and nerve fibers in both the myenteric (G) and submucosal (H) plexuses. TRPC6-IR nerve cell bodies were colocalized with Hu-IR (G’, H’). Preadsorption with the corresponding blocking peptide decreased or abolished immunostaining as shown in the example for the submucosal plexus (I-L). BP, blocking peptide. Scale bar = 20 μm for all panels. Magenta-green copy of this figure is available as supplementary figure 2.

Article Snippet: The sequence is H-RSESIAFIEESKKD ADEVFSS-NH2 Sheep 1:1,000 Chemicon/AB1529/0703053959 NPY Synthetic NPY peptide conjugated to bovine thyroglobulin Sheep 1:5,000 Chemicon/AB1583/0703053959 Synaptophysin Protein p38 6 from crude fractions of coated vesicles from bovine brain Mouse 1:200 Dako/M0776/00017473 TRPC1 Peptide QLYDK GYTSK EQKDC, corresponding to amino acid residues 557−571 of human TRPC1 (Accession {"type":"entrez-protein","attrs":{"text":"P48995","term_id":"1351302","term_text":"P48995"}} P48995 ) Rabbit 1:100 Alomone/ACC-010/AN-04 TRPC3 Peptide HKLSE KLNPS VLRC, corresponding to residues 822−835 of mouse TRPC3 (Accession {"type":"entrez-protein","attrs":{"text":"Q9QZC1","term_id":"10720320","term_text":"Q9QZC1"}} Q9QZC1 ) Rabbit 1:100 Alomone/ACC-016/AN-03 TRPC4 Peptide (C)KEKH AHEED SSIDY DL, corresponding to residues 943−958 of mouse TRPC4 (Accession {"type":"entrez-protein","attrs":{"text":"Q9QUQ5","term_id":"14548291","term_text":"Q9QUQ5"}} Q9QUQ5 ) Rabbit 1:100 Alomone/ACC-018/AN-01 TRPC5 Peptide (C)HKWGDGQEEQVTTRL corresponding to residues 959−973 of human TRPC5 (Accession {"type":"entrez-protein","attrs":{"text":"Q9UL62","term_id":"10720321","term_text":"Q9UL62"}} Q9UL62 ) Rabbit 1:100 Alomone/ACC-020/AN-07 TRPC6 Peptide (C)RRNE SQDYL LMDELG, corresponding to residues 24−38 of mouse TRPC6 (Accession {"type":"entrez-protein","attrs":{"text":"Q61143","term_id":"342187122","term_text":"Q61143"}} Q61143 ) Rabbit 1:100 Alomone/ACC-017/AN-02 TRPC7 Peptide ILKEL SKEEE DTDSS EEMLA, corresponding to residues 658−677 of human TRPC7 (Accession {"type":"entrez-nucleotide","attrs":{"text":"NM_020389","term_id":"262399373","term_text":"NM_020389"}} NM_020389 ) Rabbit 1:100 Chemicon/AB9326/VR1377347 VIP Synthetic peptide (1−28) coupled to KLH with glutaraldehyde Sheep 1:200 Chemicon/AB1581/0611044826 Open in a separate window Hu, anti-human neuronal protein; ChAT, choline-acetyltransferase; NeuN; neuronal nuclear protein; NOS; nitric oxide synthase; NPY, neuropeptide Y; TRPC, canonical transient receptor potential channel; VIP, vasoactive inhibitory peptide.

Techniques: Immunohistochemical staining, Labeling, Marker, Blocking Assay, Immunostaining

Journal:

Article Title: Differential Expression of Canonical (Classical) Transient Receptor Potential Channels in Guinea Pig Enteric Nervous System

doi: 10.1002/cne.21874

Figure Lengend Snippet: Canonical transient receptor potential channel (TRPC) mRNA expression in the guinea pig enteric nervous system (ENS). RT-PCR analysis with primers specific for each of the TRPC subunits identified mRNA transcripts for TRPC1 through TRPC7 in the guinea pig brain (i.e., positive controls). Transcripts encoding TRPC1/3/4/6/7 were expressed in the submucosal plexus (SMP). Bands representing TRPC2/5 were not detected in the SMP preparations. Transcripts encoding TRPC1 to TRPC7 were all observed in the longitudinal muscle-myenteric plexus (LMMP) preparations, while transcripts encoding TRPC1 to TRPC6 were detected also in dissociated myenteric ganglia. Sizes of the bands matched the expected lengths of the amplified fragments of each TRPC subunit (TRPC1, 400 bp; TRPC2, 370 bp; TRPC3, 340 bp; TRPC4, 370 bp; TRPC5, 484 bp; TRPC6 327 bp; TRPC7 429 bp).

Article Snippet: The sequence is H-RSESIAFIEESKKD ADEVFSS-NH2 Sheep 1:1,000 Chemicon/AB1529/0703053959 NPY Synthetic NPY peptide conjugated to bovine thyroglobulin Sheep 1:5,000 Chemicon/AB1583/0703053959 Synaptophysin Protein p38 6 from crude fractions of coated vesicles from bovine brain Mouse 1:200 Dako/M0776/00017473 TRPC1 Peptide QLYDK GYTSK EQKDC, corresponding to amino acid residues 557−571 of human TRPC1 (Accession {"type":"entrez-protein","attrs":{"text":"P48995","term_id":"1351302","term_text":"P48995"}} P48995 ) Rabbit 1:100 Alomone/ACC-010/AN-04 TRPC3 Peptide HKLSE KLNPS VLRC, corresponding to residues 822−835 of mouse TRPC3 (Accession {"type":"entrez-protein","attrs":{"text":"Q9QZC1","term_id":"10720320","term_text":"Q9QZC1"}} Q9QZC1 ) Rabbit 1:100 Alomone/ACC-016/AN-03 TRPC4 Peptide (C)KEKH AHEED SSIDY DL, corresponding to residues 943−958 of mouse TRPC4 (Accession {"type":"entrez-protein","attrs":{"text":"Q9QUQ5","term_id":"14548291","term_text":"Q9QUQ5"}} Q9QUQ5 ) Rabbit 1:100 Alomone/ACC-018/AN-01 TRPC5 Peptide (C)HKWGDGQEEQVTTRL corresponding to residues 959−973 of human TRPC5 (Accession {"type":"entrez-protein","attrs":{"text":"Q9UL62","term_id":"10720321","term_text":"Q9UL62"}} Q9UL62 ) Rabbit 1:100 Alomone/ACC-020/AN-07 TRPC6 Peptide (C)RRNE SQDYL LMDELG, corresponding to residues 24−38 of mouse TRPC6 (Accession {"type":"entrez-protein","attrs":{"text":"Q61143","term_id":"342187122","term_text":"Q61143"}} Q61143 ) Rabbit 1:100 Alomone/ACC-017/AN-02 TRPC7 Peptide ILKEL SKEEE DTDSS EEMLA, corresponding to residues 658−677 of human TRPC7 (Accession {"type":"entrez-nucleotide","attrs":{"text":"NM_020389","term_id":"262399373","term_text":"NM_020389"}} NM_020389 ) Rabbit 1:100 Chemicon/AB9326/VR1377347 VIP Synthetic peptide (1−28) coupled to KLH with glutaraldehyde Sheep 1:200 Chemicon/AB1581/0611044826 Open in a separate window Hu, anti-human neuronal protein; ChAT, choline-acetyltransferase; NeuN; neuronal nuclear protein; NOS; nitric oxide synthase; NPY, neuropeptide Y; TRPC, canonical transient receptor potential channel; VIP, vasoactive inhibitory peptide.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification

Journal:

Article Title: Differential Expression of Canonical (Classical) Transient Receptor Potential Channels in Guinea Pig Enteric Nervous System

doi: 10.1002/cne.21874

Figure Lengend Snippet: TRPC3-IR nerve fibers in the submucosal plexus. A: TRPC3-IR varicose nerve fibers were observed in the tertiary division of the submucosal plexus. B: TRPC3-IR varicose nerve fibers were seen surrounding submucosal blood vessels. C-D: Paired images demonstrating colocalization of TRPC3-IR with IR for the presynaptic vesicular protein, synaptophysin. The arrows in C and D point to nerve fibers that express TRPC3-IR and synaptophysin-IR. TRPC3-IR was expressed at some, but not all, varicosities. Scale bar = 20 μm for all panels.

Article Snippet: The sequence is H-RSESIAFIEESKKD ADEVFSS-NH2 Sheep 1:1,000 Chemicon/AB1529/0703053959 NPY Synthetic NPY peptide conjugated to bovine thyroglobulin Sheep 1:5,000 Chemicon/AB1583/0703053959 Synaptophysin Protein p38 6 from crude fractions of coated vesicles from bovine brain Mouse 1:200 Dako/M0776/00017473 TRPC1 Peptide QLYDK GYTSK EQKDC, corresponding to amino acid residues 557−571 of human TRPC1 (Accession {"type":"entrez-protein","attrs":{"text":"P48995","term_id":"1351302","term_text":"P48995"}} P48995 ) Rabbit 1:100 Alomone/ACC-010/AN-04 TRPC3 Peptide HKLSE KLNPS VLRC, corresponding to residues 822−835 of mouse TRPC3 (Accession {"type":"entrez-protein","attrs":{"text":"Q9QZC1","term_id":"10720320","term_text":"Q9QZC1"}} Q9QZC1 ) Rabbit 1:100 Alomone/ACC-016/AN-03 TRPC4 Peptide (C)KEKH AHEED SSIDY DL, corresponding to residues 943−958 of mouse TRPC4 (Accession {"type":"entrez-protein","attrs":{"text":"Q9QUQ5","term_id":"14548291","term_text":"Q9QUQ5"}} Q9QUQ5 ) Rabbit 1:100 Alomone/ACC-018/AN-01 TRPC5 Peptide (C)HKWGDGQEEQVTTRL corresponding to residues 959−973 of human TRPC5 (Accession {"type":"entrez-protein","attrs":{"text":"Q9UL62","term_id":"10720321","term_text":"Q9UL62"}} Q9UL62 ) Rabbit 1:100 Alomone/ACC-020/AN-07 TRPC6 Peptide (C)RRNE SQDYL LMDELG, corresponding to residues 24−38 of mouse TRPC6 (Accession {"type":"entrez-protein","attrs":{"text":"Q61143","term_id":"342187122","term_text":"Q61143"}} Q61143 ) Rabbit 1:100 Alomone/ACC-017/AN-02 TRPC7 Peptide ILKEL SKEEE DTDSS EEMLA, corresponding to residues 658−677 of human TRPC7 (Accession {"type":"entrez-nucleotide","attrs":{"text":"NM_020389","term_id":"262399373","term_text":"NM_020389"}} NM_020389 ) Rabbit 1:100 Chemicon/AB9326/VR1377347 VIP Synthetic peptide (1−28) coupled to KLH with glutaraldehyde Sheep 1:200 Chemicon/AB1581/0611044826 Open in a separate window Hu, anti-human neuronal protein; ChAT, choline-acetyltransferase; NeuN; neuronal nuclear protein; NOS; nitric oxide synthase; NPY, neuropeptide Y; TRPC, canonical transient receptor potential channel; VIP, vasoactive inhibitory peptide.

Techniques:

Journal:

Article Title: Differential Expression of Canonical (Classical) Transient Receptor Potential Channels in Guinea Pig Enteric Nervous System

doi: 10.1002/cne.21874

Figure Lengend Snippet: Neurochemical coding of TRPC3-IR in the submucosal plexus. TRPC3- IR was colocalized with ChAT (A, A’) and NPY (B, B’). No colocalization of TRPC3 with calretinin (C, C’), VIP (D, D’), calbindin (E, E’), nor NeuN (F, F’) was found. Arrows indicate neurons that are positive for TRPC3 as well as neurochemical markers. Arrowheads indicate neurons that are positive for TRPC3 only. Scale bar = 20 μm for all panels.

Article Snippet: The sequence is H-RSESIAFIEESKKD ADEVFSS-NH2 Sheep 1:1,000 Chemicon/AB1529/0703053959 NPY Synthetic NPY peptide conjugated to bovine thyroglobulin Sheep 1:5,000 Chemicon/AB1583/0703053959 Synaptophysin Protein p38 6 from crude fractions of coated vesicles from bovine brain Mouse 1:200 Dako/M0776/00017473 TRPC1 Peptide QLYDK GYTSK EQKDC, corresponding to amino acid residues 557−571 of human TRPC1 (Accession {"type":"entrez-protein","attrs":{"text":"P48995","term_id":"1351302","term_text":"P48995"}} P48995 ) Rabbit 1:100 Alomone/ACC-010/AN-04 TRPC3 Peptide HKLSE KLNPS VLRC, corresponding to residues 822−835 of mouse TRPC3 (Accession {"type":"entrez-protein","attrs":{"text":"Q9QZC1","term_id":"10720320","term_text":"Q9QZC1"}} Q9QZC1 ) Rabbit 1:100 Alomone/ACC-016/AN-03 TRPC4 Peptide (C)KEKH AHEED SSIDY DL, corresponding to residues 943−958 of mouse TRPC4 (Accession {"type":"entrez-protein","attrs":{"text":"Q9QUQ5","term_id":"14548291","term_text":"Q9QUQ5"}} Q9QUQ5 ) Rabbit 1:100 Alomone/ACC-018/AN-01 TRPC5 Peptide (C)HKWGDGQEEQVTTRL corresponding to residues 959−973 of human TRPC5 (Accession {"type":"entrez-protein","attrs":{"text":"Q9UL62","term_id":"10720321","term_text":"Q9UL62"}} Q9UL62 ) Rabbit 1:100 Alomone/ACC-020/AN-07 TRPC6 Peptide (C)RRNE SQDYL LMDELG, corresponding to residues 24−38 of mouse TRPC6 (Accession {"type":"entrez-protein","attrs":{"text":"Q61143","term_id":"342187122","term_text":"Q61143"}} Q61143 ) Rabbit 1:100 Alomone/ACC-017/AN-02 TRPC7 Peptide ILKEL SKEEE DTDSS EEMLA, corresponding to residues 658−677 of human TRPC7 (Accession {"type":"entrez-nucleotide","attrs":{"text":"NM_020389","term_id":"262399373","term_text":"NM_020389"}} NM_020389 ) Rabbit 1:100 Chemicon/AB9326/VR1377347 VIP Synthetic peptide (1−28) coupled to KLH with glutaraldehyde Sheep 1:200 Chemicon/AB1581/0611044826 Open in a separate window Hu, anti-human neuronal protein; ChAT, choline-acetyltransferase; NeuN; neuronal nuclear protein; NOS; nitric oxide synthase; NPY, neuropeptide Y; TRPC, canonical transient receptor potential channel; VIP, vasoactive inhibitory peptide.

Techniques:

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

Article Title: Ca 2+ -binding protein 2 inhibits Ca 2+ -channel inactivation in mouse inner hair cells

doi: 10.1073/pnas.1617533114

Figure Lengend Snippet: Normal number and gross molecular anatomy of IHCs and their ribbon synapses in Cabp2LacZ/LacZ mice. (A) Representative maximum projection of immunostaining for large-conductance Ca2+-activated K+ channels (BK, magenta) and parvalbumin-α (Pavalb., green) of mice at the age of P15. BK channels are clustered in the neck region of Cabp2LacZ/LacZ and Cabp2+/+ IHCs, indicating normal development. (Scale bars: 10 μm.) (B) Representative maximum projection of immunostaining for small-conductance Ca2+-activated K+ channels (SK2, magenta) and calretinin (Calret., green) of Cabp2LacZ/LacZ and Cabp2+/+ IHCs showing that SK2 puncta are restricted to OHCs and lacking in IHCs. The white arrowheads indicate SK2 channel puncta in OHCs. (Scale bars: 10 μm.) (C) Representative synapse immunostaining of Cabp2+/+ and Cabp2LacZ/LacZ IHCs of P21–P28 animals. Ribbon-occupied synapses were identified by colocalized presynaptic ribbon (RIBEYE/Ctbp2, magenta) and a postsynaptic bouton (GluA2/3, green). (Scale bars: 10 μm.) (D) Same experimental design as in C was also applied to organs of Corti of P56–P63 mice. (Scale bars: 10 μm.) (E, Left) Ribbon-occupied synapses are identified as juxtaposed spots of RIBEYE/Ctbp2 and GluA2/3 immunofluorescence (schematic with one exemplary synapse drawn). (E, Right) Quantification of ribbon-occupied synapses/IHC for Cabp2+/+ and Cabp2LacZ/LacZ IHCs of both age groups: No significant differences (n.s.) in synapse numbers were detected between genotypes of the respective age (3 wk: Wilcoxon rank sum test; 8 wk: Student’s t test). For all four conditions, organs of Corti of four animals were used. (F) Representative immunostaining of Ca2+ channels (CaV1.3, green) and the presynaptic ribbon (RIBEYE/Ctbp2, magenta) showing proper cluster formation of CaV1.3 at the active zone for Cabp2LacZ/LacZ and Cabp2+/+ IHCs. (Scale bars: 10 μm.)

Article Snippet: The following antibodies were used: guinea pig anti–parvalbumin-α (1:200; Synaptic Systems), rabbit anti-BK (1:100; Alomone), mouse anti–α-calbindin D-28k (1:400; SWANT), rabbit anti-SK2 (1:200; Alomone), mouse IgG1 anti-CtBP2 (1:200; BD Biosciences, rabbit anti-GluA2/3 (1:200;Chemicon), and rabbit–anti-Ca V 1.3 (1:100; Alomone).

Techniques: Immunostaining, Immunofluorescence

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Sound Rhythms Are Encoded by Postinhibitory Rebound Spiking in the Superior Paraolivary Nucleus

doi: 10.1523/JNEUROSCI.2450-11.2011

Figure Lengend Snippet: SPON neurons express HCN1 and HCN2 channels and have large Ih inwardly rectifying (IR) currents. A, HCN1 immunoreactivity in the superior olivary complex of an adult mouse. The low-magnification micrograph illustrates that the lateral superior olive expresses the strongest HCN1-like immunostaining, the SPON displays moderate levels of expression, and the MNTB shows the weakest immunoreactivity. At higher magnification, it is evident that both somata (arrowheads) and dendrites (arrow) of SPON neurons are immunopositive. Asterisks denote landmarks in slice. Scale bars: left, 200 μm; right, 50 μm. B) HCN2 is most strongly expressed in the MNTB, whereas expression in the LSO is weakest. At higher magnification, it is evident that SPON neurons are also immunopositive. Asterisks denote landmarks in slice. Scale bars: left, 200 μm; right, 50 μm. C, Current traces recorded from an SPON neuron induced by hyperpolarization from a holding current of −62 to −122 mV, in −10 mV voltage steps, under control conditions (top) and during pharmacological blockade of Ih with 20 μM ZD7288 (bottom). D, The size of the IR current was measured at steady state (i.e., ~1.35 s after induction) at each hyperpolarizing voltage (n = 42). The IR currents were significantly diminished by ZD7288 (n = 6) in the SPON neurons. ***p < 0.001, Student’s t test. E, Average activation time constants of the IR currents in SPON neurons were obtained by fitting a single-exponential function to the current traces.

Article Snippet: Sections were incubated in 2% normal donkey serum in blocking solution overnight at 4°C with one of the following primary antibodies: polyclonal rabbit α -HCN1 (1:250, lot number AN-10; Alomone Labs), which is directed against amino acid residues 6–24 of the intracellular N terminus of rat HCN1 (GenBank accession number {"type":"entrez-protein","attrs":{"text":"Q9JKB0","term_id":"29840774","term_text":"Q9JKB0"}} Q9JKB0 ); polyclonal rabbit α -HCN2 (1:400, lot number AN-08; Alomone Labs), directed against amino acids 147–161 of the intracellular N terminus of human HCN2 (GenBank accession number {"type":"entrez-protein","attrs":{"text":"Q9UL51","term_id":"108935843","term_text":"Q9UL51"}} Q9UL51 ); or monoclonal mouse α -HCN2 (1:400, clone N71/37; NeuroMab, UC Davis/NIH NeuroMab Facility) directed against amino acids 761–863 of the C terminus of rat HCN2 (GenBank accession number {"type":"entrez-protein","attrs":{"text":"Q9JKA9","term_id":"83303515","term_text":"Q9JKA9"}} Q9JKA9 ) used in combination with a mouse-on-mouse kit (Vector Laboratories).

Techniques: Immunostaining, Expressing, Activation Assay