Journal: Nature Communications

Article Title: TRPC6 specifically interacts with APP to inhibit its cleavage by γ-secretase and reduce Aβ production

doi: 10.1038/ncomms9876

Figure Lengend Snippet: ( a ) Left: ELISA examination of Aβ levels in the medium of primary cultured cortical neurons transfected with the indicated siRNAs ( n =3–4 in duplication). Immunoblot analysis (middle) and quantification (right) of the expression levels of the indicated proteins in the neurons transfected with siRNAs ( n =3–4 in duplication). ( b ) Aβ levels in the medium of the cortical neurons treated with OAG (50 μM) or SKF96365 (2 μM) for 12 h ( n =3). ( c ) Aβ levels in the medium of HEK293APP stable cells transfected with TRPC5 or 6 for 2 days ( n =5 in duplication). ( d ) Aβ levels in the medium of HEK293APP stable cells transfected with YFP or TRPC6 for 1 day and then treated with vehicle or SKF96365 (1 μM) for 12 h. SKF96365 did not suppress TRPC6 inhibition of Aβ levels ( n =4). ( e ) Aβ levels in the medium of HEK293APP cells transfected with indicated constructs for 2 days ( n =4–5). Insets, representative immunobloting of indicated proteins. NC, negative control siRNA. CTRL, vehicle or transfection with YFP . Data were presented as means±s.e.m. of indicated numbers of independent experiments. One way ANOVA with Newman–Keuls post hoc test was performed. * P <0.05, ** P <0.01, *** P <0.001 versus NC or CTRL.

Article Snippet: The following antibodies were used: TRPC6 from Alomone (ACC-017) , Sigma (PRS3889) and SAB (21403), TRPC5 from Sigma (ACC-020) , Ca v 1.2 from Millipore (AB5156), Ca v 3.1 (ACC-021) and Ca v 3.3 (ACC-009) from Alomone, APP from Invitrogen (13-0200, clone LN27) and Sigma (A8717), 6E10 from Chemicon (MAB1560) and Signet (SIG-39300), MOAB-2 from Kerafast (EB2001), ADAM10 from Abcam (Ab1997), clusterin from Santa Cruz (sc-6420), BACE1 from Millipore (MAB5308), insulin-degrading enzyme from EMD Biosciences (PC730), neprilysin from Epitomics (2569–1), PS1 from Chemicon (MAB5232), nicastrin from Sigma (MAB5232), presenilin enhancer 2 from Invitrogen (36–7100), E-Cadherin from Abcam (ab53033), N-Cadherin from BD (610921), cleaved-Notch from Cell Signalling Technology (2421 L), Notch from Proteintech (10062-2-AP), EEA1 from BD (610457), Calnexin from Enzo (ADI-SPA-860-F), GM130 from BD (610823), Bip from BD (610979), Cathepsin D from Santa Cruz (sc-6487), Myc from Chemicon (05–724), HA from Sigma (H6908) and Genscript (A00168), and His from Abmart (M30111).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Transfection, Western Blot, Expressing, Inhibition, Construct, Negative Control

Journal: Nature Communications

Article Title: TRPC6 specifically interacts with APP to inhibit its cleavage by γ-secretase and reduce Aβ production

doi: 10.1038/ncomms9876

Figure Lengend Snippet: ( a ) Aβ levels in the medium of COS7C99 stable cells transfected with YFP or TRPC6 for 2 days ( n =3–4). ( b ) Immunoblot analysis of AICD in HEK293C99 cells transfected with YFP or TRPC6 for 2 days. ( c ) Quantification of the ratio of AICD/C99 ( n =3). ( d ) Immunoblots of presenilin 1 (PS1), PS2, nicastrin (Nct) and presenilin enhance 2 (Pen2) in HEK293APP cells transfected with TRPC6 for 2 days. ( e ) γ-Secretase activity in the in vitro fluorogenic substrate assay of HEK293APP cells transfected with TRPC6 for 2 days ( n =3–5). ( f ) Immunoblot analysis of Notch intracellular domain (NICD) in HEK293APP cells transfected with NotchΔE-myc for 1 day and then further transfected with YFP or TRPC6 for 2 days. ( g ) Quantification of the ratio of NICD/NotchΔE ( n =4). ( h ) Immunoblots of CTF1 and CTF2 of E-/N-Cadherin in HEK293APP cells transfected with YFP or TRPC6 for 2 days. ( i ) Quantification of CTF2/CTF1 of E-/N-Cadherin ( n =3–4). CTRL, transfection with YFP or pcDNA3.1. L658,458: γ-secretase inhibitor. Data were presented as means±s.e.m. of indicated numbers of independent experiments. Two-tailed Student's t -test was performed. * P <0.05, ** P <0.01 versus CTRL.

Article Snippet: The following antibodies were used: TRPC6 from Alomone (ACC-017) , Sigma (PRS3889) and SAB (21403), TRPC5 from Sigma (ACC-020) , Ca v 1.2 from Millipore (AB5156), Ca v 3.1 (ACC-021) and Ca v 3.3 (ACC-009) from Alomone, APP from Invitrogen (13-0200, clone LN27) and Sigma (A8717), 6E10 from Chemicon (MAB1560) and Signet (SIG-39300), MOAB-2 from Kerafast (EB2001), ADAM10 from Abcam (Ab1997), clusterin from Santa Cruz (sc-6420), BACE1 from Millipore (MAB5308), insulin-degrading enzyme from EMD Biosciences (PC730), neprilysin from Epitomics (2569–1), PS1 from Chemicon (MAB5232), nicastrin from Sigma (MAB5232), presenilin enhancer 2 from Invitrogen (36–7100), E-Cadherin from Abcam (ab53033), N-Cadherin from BD (610921), cleaved-Notch from Cell Signalling Technology (2421 L), Notch from Proteintech (10062-2-AP), EEA1 from BD (610457), Calnexin from Enzo (ADI-SPA-860-F), GM130 from BD (610823), Bip from BD (610979), Cathepsin D from Santa Cruz (sc-6487), Myc from Chemicon (05–724), HA from Sigma (H6908) and Genscript (A00168), and His from Abmart (M30111).

Techniques: Transfection, Western Blot, Activity Assay, In Vitro, Two Tailed Test

Journal: Nature Communications

Article Title: TRPC6 specifically interacts with APP to inhibit its cleavage by γ-secretase and reduce Aβ production

doi: 10.1038/ncomms9876

Figure Lengend Snippet: ( a ) Immunocytochemical analysis of HEK293 cells transfected with APP-Myc and TRPC6-HA for 3 days. EEA1, Calnexin and GM130 are markers for early endosome, ER and Golgi, respectively. Arrow heads indicated the co-localized signals. Scale bar, 20 μm. ( b ) Immunoblots of indicated proteins after fractionation of mouse brain lysates. Bip and Cathepsin D are markers for ER and lysosome, respectively. Arrow indicated alignment of two gels with 11 lanes running in parallel. ( c ) Wild-type mouse brain lysates precipitated with the antibody against APP, PS1 or Notch, and immunoblotted with indicated antibodies. ( d ) Lysates of HEK293APP stable cells transfected with TRPC5 or 6 for 2 days precipitated with APP antibody, and immunoblotted with indicated antibodies. ( e ) Lysates of HEK293TRPC6 stable cells transfected with C99-Myc or NotchΔE-Myc for 1 day, treated with 10 μM L685,458 for 12 h and then precipitated with Myc or TRPC6 antibody, and immunoblotted with indicated antibodies. ( f ) Lysates of HEK293C99-Myc stable cells transfected with PS1-HA together with YFP or TRPC6 for 2 days and precipitated with Myc or HA antibody, and immunoblotted with indicated antibodies. Lower, quantification of the reciprocal precipitated protein levels of C99 and PS1 ( n =4). CTRL, transfection with YFP. Data were presented as means±s.e.m. of indicated numbers of independent experiments. Two-tailed Student's t -test was performed. * P <0.05, ** P <0.01 versus CTRL.

Article Snippet: The following antibodies were used: TRPC6 from Alomone (ACC-017) , Sigma (PRS3889) and SAB (21403), TRPC5 from Sigma (ACC-020) , Ca v 1.2 from Millipore (AB5156), Ca v 3.1 (ACC-021) and Ca v 3.3 (ACC-009) from Alomone, APP from Invitrogen (13-0200, clone LN27) and Sigma (A8717), 6E10 from Chemicon (MAB1560) and Signet (SIG-39300), MOAB-2 from Kerafast (EB2001), ADAM10 from Abcam (Ab1997), clusterin from Santa Cruz (sc-6420), BACE1 from Millipore (MAB5308), insulin-degrading enzyme from EMD Biosciences (PC730), neprilysin from Epitomics (2569–1), PS1 from Chemicon (MAB5232), nicastrin from Sigma (MAB5232), presenilin enhancer 2 from Invitrogen (36–7100), E-Cadherin from Abcam (ab53033), N-Cadherin from BD (610921), cleaved-Notch from Cell Signalling Technology (2421 L), Notch from Proteintech (10062-2-AP), EEA1 from BD (610457), Calnexin from Enzo (ADI-SPA-860-F), GM130 from BD (610823), Bip from BD (610979), Cathepsin D from Santa Cruz (sc-6487), Myc from Chemicon (05–724), HA from Sigma (H6908) and Genscript (A00168), and His from Abmart (M30111).

Techniques: Transfection, Western Blot, Fractionation, Two Tailed Test

Journal: Nature Communications

Article Title: TRPC6 specifically interacts with APP to inhibit its cleavage by γ-secretase and reduce Aβ production

doi: 10.1038/ncomms9876

Figure Lengend Snippet: ( a ) ELISA examination of Aβ levels in the medium of HEK293APP cells transfected with TRPC6 or its mutants within second transmembrane domain (TM2) for 2 days ( n =3–6). ( b ) Lysates of HEK293APP stable cells transfected with C6-mut1 for 2 days precipitated with APP antibody and immunoblotted with indicated antibodies. ( c ) The Aβ levels in the medium of HEK293APP cells treated with 2 μM indicated peptides for 12 h ( n =3–6). ( d ) Lysates of HEK293APP stable cells incubated with 5 μM TAT-biotin or TAT-TM2-bitoin for 6 h precipitated with avidin beads and immunoblotted with indicated antibodies. ( e ) ELISA examination in a blinded manner of Aβ levels in the brain lysates of 5 month male APP/PS1 mice intra-peritoneally injected with 200 μl 2.5 mM TAT or TAT-TM2 for 3 h ( n =8 mice). ( f ) The Aβ levels generated in the in vitro purified C99 cleavage assay treated with 2 μM of indicated peptides for 2 h ( n =4). ( g ) Aβ levels in the medium of COS7C99 stable cells treated with 5 μM TAT-TM2 for 12 h ( n =3–4). ( h ) Immunoblots of AICD in HEK293C99 cells treated with 5 μM TAT-TM2 for 12 h or NICD in HEK293APP cells transfected with Notch≜E-myc for 1 day and then treated with 5 μM TAT-TM2 for 12 h. ( i ) Quantification of the ratio of AICD/C99 or NICD/NotchΔE ( n =3–4). CTRL, transfection with YFP . Data were presented as means±s.e.m. of indicated numbers of independent experiments. Two-tailed Student's t -test was performed for two groups, and one way ANOVA with Newman–Keuls post hoc test was performed for more than two groups. * P <0.05, *** P <0.001 versus CTRL or TAT.

Article Snippet: The following antibodies were used: TRPC6 from Alomone (ACC-017) , Sigma (PRS3889) and SAB (21403), TRPC5 from Sigma (ACC-020) , Ca v 1.2 from Millipore (AB5156), Ca v 3.1 (ACC-021) and Ca v 3.3 (ACC-009) from Alomone, APP from Invitrogen (13-0200, clone LN27) and Sigma (A8717), 6E10 from Chemicon (MAB1560) and Signet (SIG-39300), MOAB-2 from Kerafast (EB2001), ADAM10 from Abcam (Ab1997), clusterin from Santa Cruz (sc-6420), BACE1 from Millipore (MAB5308), insulin-degrading enzyme from EMD Biosciences (PC730), neprilysin from Epitomics (2569–1), PS1 from Chemicon (MAB5232), nicastrin from Sigma (MAB5232), presenilin enhancer 2 from Invitrogen (36–7100), E-Cadherin from Abcam (ab53033), N-Cadherin from BD (610921), cleaved-Notch from Cell Signalling Technology (2421 L), Notch from Proteintech (10062-2-AP), EEA1 from BD (610457), Calnexin from Enzo (ADI-SPA-860-F), GM130 from BD (610823), Bip from BD (610979), Cathepsin D from Santa Cruz (sc-6487), Myc from Chemicon (05–724), HA from Sigma (H6908) and Genscript (A00168), and His from Abmart (M30111).

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Incubation, Avidin-Biotin Assay, Injection, Generated, In Vitro, Purification, Cleavage Assay, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: TRPC6 specifically interacts with APP to inhibit its cleavage by γ-secretase and reduce Aβ production

doi: 10.1038/ncomms9876

Figure Lengend Snippet: ( a ) Representative images of amyloid plaque immunostained with Aβ antibody (6E10) on brain sections from 6 months female APP/PS1 and APP/PS1/TRPC6 mice. Scale bar, 500 μm. ( b ) Quantification of the plaque load in indicated mouse brain sections shown in a ( n =8). ELISA of total ( c ) TBST soluble ( d ) or insoluble ( e ) Aβ levels in the forebrain lysates of 6 month female indicated mice ( n =8). Representative images ( f ) and quantification ( g ) of thioflavin S staining of amyloid plaque in the hippocampal region of 6 and 11 months APP/PS1 and APP/PS1/TRPC6 mice with B6C3 background ( n =4–5 mice). Scale bar, 200 μm. Data were presented as means±s.e.m. Two-tailed Student's t -test was performed. * P <0.05, ** P <0.01 versus APP/PS1 .

Article Snippet: The following antibodies were used: TRPC6 from Alomone (ACC-017) , Sigma (PRS3889) and SAB (21403), TRPC5 from Sigma (ACC-020) , Ca v 1.2 from Millipore (AB5156), Ca v 3.1 (ACC-021) and Ca v 3.3 (ACC-009) from Alomone, APP from Invitrogen (13-0200, clone LN27) and Sigma (A8717), 6E10 from Chemicon (MAB1560) and Signet (SIG-39300), MOAB-2 from Kerafast (EB2001), ADAM10 from Abcam (Ab1997), clusterin from Santa Cruz (sc-6420), BACE1 from Millipore (MAB5308), insulin-degrading enzyme from EMD Biosciences (PC730), neprilysin from Epitomics (2569–1), PS1 from Chemicon (MAB5232), nicastrin from Sigma (MAB5232), presenilin enhancer 2 from Invitrogen (36–7100), E-Cadherin from Abcam (ab53033), N-Cadherin from BD (610921), cleaved-Notch from Cell Signalling Technology (2421 L), Notch from Proteintech (10062-2-AP), EEA1 from BD (610457), Calnexin from Enzo (ADI-SPA-860-F), GM130 from BD (610823), Bip from BD (610979), Cathepsin D from Santa Cruz (sc-6487), Myc from Chemicon (05–724), HA from Sigma (H6908) and Genscript (A00168), and His from Abmart (M30111).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Two Tailed Test

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: TRPC6-IR nerve fibers in myenteric and submucosal plexuses. A: TRPC6-IR varicose nerve fibers in myenteric ganglia and in interganglionic fiber bundles. Within some myenteric ganglia, TRPC6-IR fibers form complexes that encircle individual ganglionic cell somas. B: TRPC6-IR nerve fibers were present in the tertiary division of the myenteric plexus. C: TRPC6-IR nerve cell bodies and varicose nerve fibers in submucosal ganglia and in interganglionic connectives. D: TRPC6-IR nerve fibers in the tertiary division of the submucosal plexus. E: TRPC6-IR nerve fibers in the circular muscle plexus. F: TRPC6-IR nerve fibers around submucosal blood vessels. G-H: Paired images showing colocalization of TRPC6-IR with synaptophysin-IR in the circular muscle plexus. The arrows point to nerve fibers that coexpress TRPC6- and synaptophysin-IR. 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.

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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 TRPC6-immunoreactive neurons in the myenteric plexus of the guinea pig ileum. TRPC6 IR was colocalized with ChAT (A, A’) and NOS (C, C’), but was not colocalized with calretinin (B, B’) nor calbindin (D, D’). Arrows indicate neurons that are positive for TRPC6 as well as neurochemical markers. Arrowheads indicate neurons that are positive for TRPC6 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:

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 TRPC6-IR in the submucosal plexus. TRPC6-IR was exclusively colocalized with VIP (D, D’). No colocalization of TRPC6 with ChAT (A, A’), calretinin (B, B’), NPY (C, C’), calbindin (E, E’) nor NeuN (F, F’) was found. Arrows indicate neurons that are positive for TRPC6 as well as neurochemical markers. Arrowheads indicate neurons that are positive for TRPC6 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: