Journal: Scientific Reports

Article Title: The Na +/ H + -Exchanger NHE1 Regulates Extra- and Intracellular pH and Nimodipine-sensitive [Ca 2+ ] i in the Suprachiasmatic Nucleus

doi: 10.1038/s41598-019-42872-w

Figure Lengend Snippet: NHE1 colocalisation with CaV1.2 ( A ) and NCX1 ( B ), as well as colocalisation of NHE1/CaV1.2 with SERT ( C ), NPY ( D ), and Bassoon ( E ). ( A , B ) High magnification images showing high levels of NHE1 colocalisation with CaV1.2 ( A ), but not NCX1 ( B ). ( C – E ) High magnification images showing moderate to high levels of NHE1/CaV1.2 colocalisation with SERT ( C ) and Bassoon ( E ), but not NPY ( D ). Scale bar: 10 µm.

Article Snippet: For immunofluorescence staining, sections (20 µm) were washed for 20–30 min in PBS and then incubated overnight at 4 °C in PBS containing 2% serum, 0.3% Triton X-100, and primary antibodies against NHE1 (rabbit anti-NHE1; 1:100; ab67314, RRID:AB_1141782; Abcam, Cambridge, MA, USA), neurophysin II (NP2) (goat anti-NP2; 1:500; sc-27093, RRID:AB_2061964; Santa Cruz, CA, USA), vasoactive intestinal peptide (VIP) (guinea pig anti-VIP; 1:500; T-5030, RRID:AB_518690; Peninsula Laboratories, San Carlos, CA, USA), gastrin-releasing peptide (GRP) (goat anti-GRP; 1:100; sc-7788, RRID:AB_2232721; Santa Cruz, CA, USA), vesicular glutamate transporter type 2 (vGluT2) (guinea pig anti-vGluT2; 1:300; AB2251, RRID:AB_1587626; Millipore, Temecula, CA, USA), serotonin transporter (SERT) (mouse anti-SERT; 1:200; MAB1564, RRID:AB_94220; Millipore, Temecula, CA, USA), neuropeptide Y (NPY) (goat anti-NPY; 1:300; NBP1-46535, RRID:AB_10009813; Novus, Littleton, CO, USA), CaV1.2 (guinea pig anti-CaV1.2; 1:100; AGP-001; RRID:AB_11219156; Alomone Labs, Jerusalem, Israel), NCX1 (mouse anti-NCX1, against epitope between amino acid 371 and 525 on intracellular side of plasma membrane; 1:100; AB2869, RRID:AB_2191134; Abcam, MA, USA), and Bassoon (mouse anti-Bassoon; 1:200; ADI-VAM-PS003-D, RRID:AB_2038857; Enzo Life Sciences, Farmingdale, NY, USA).

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: TRPC1 is regulated by caveolin‐1 and is involved in oxidized LDL‐induced apoptosis of vascular smooth muscle cells

doi: 10.1111/j.1582-4934.2008.00593.x

Figure Lengend Snippet: TRPC1 expression is linked to caveolin‐1. TRPC1 expression is correlated with caveolin‐1 expression. (A), TRPC1 and caveolin‐1 expression were analysed in human primary VSMC (hSMC, passage 5), in SMC stably expressing caveolin‐1 cell line (SMC/cav1) and in control SMC expressing an empty vector (SMC/ev), by immunoblotting using anti‐TRPC1 and anti‐caveolin‐1 antibodies as described under ‘Materials and methods’. The graph represents values of caveolin‐1 and TRPC1 band intensity after normalization for β‐actin by densitometry (a.u., arbitrary units). Results are representative of at least three independent experiments. (*P < 0.05, **P < 0.01). (B), TRPC1 expression under caveolin‐1 silencing. Expression of TRPC1 protein was analysed by immunoblotting using an anti‐TRPC1 antibody as described under ‘Materials and methods’, after transfection of SMC/cav1 with 100 nM caveolin‐1 siRNA (siRNA cav1) for 48 and 72 hrs or 100 nM scrambled siRNA (siRNA sc). The graph represents values of caveolin‐1 and TRPC1 band intensity after normalization for β‐actin by densitometry (a.u., arbitrary units). Results are representative of at least three independent experiments. (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The following antibodies were used: polyclonal anti‐TRPC1 (Alomone Labs, Jerusalem, Israel), polyclonal anti‐caveolin‐1 and monoclonal anti‐β‐actin (Upstate Biotechnology, Billerica, MA, USA).

Techniques: Expressing, Stable Transfection, Plasmid Preparation, Western Blot, Transfection

Journal: Journal of Cellular and Molecular Medicine

Article Title: TRPC1 is regulated by caveolin‐1 and is involved in oxidized LDL‐induced apoptosis of vascular smooth muscle cells

doi: 10.1111/j.1582-4934.2008.00593.x

Figure Lengend Snippet: Caveolin‐1 silencing reduced oxLDL‐induced cell death. Caveolin‐1 silencing shifted oxLDL‐induced cell apoptosis. SMC/cav1 (A) or human primary VSMC (passage 5) (B) were either transfected with 100 nM caveolin‐1 siRNA (siRNA cav1) or with 100 nM scrambled siRNA (siRNA sc) then serum starved, and incubated with oxLDL (100 μg ApoB/ml) for 18 hrs. Expression of caveolin‐1 protein was analysed by immunoblotting using an anti‐caveolin‐1 antibody as described under ‘Materials and methods’ and cell viability was evaluated by the MTT assay. Results are expressed as percentage of untreated control and represent the mean ± S.E.M. of three independent experiments (*P < 0.05).

Article Snippet: The following antibodies were used: polyclonal anti‐TRPC1 (Alomone Labs, Jerusalem, Israel), polyclonal anti‐caveolin‐1 and monoclonal anti‐β‐actin (Upstate Biotechnology, Billerica, MA, USA).

Techniques: Transfection, Incubation, Expressing, Western Blot, MTT Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: TRPC1 is regulated by caveolin‐1 and is involved in oxidized LDL‐induced apoptosis of vascular smooth muscle cells

doi: 10.1111/j.1582-4934.2008.00593.x

Figure Lengend Snippet: OxLDL induce translocation of TRPC1 into caveolar compartment. (A), TRPC1 expression after oxLDL treatment. SMC/cav1 were incubated with oxLDL (100 μg ApoB/ml) for 10 hrs and expression of TRPC1 protein was analysed by immunoblotting using an anti‐TRPC1 antibody as described under ‘Materials and methods’. Results are representative of at least three independent experiments. (B), Increase in surface‐expressed TRPC1 induced by oxLDL. SMC/cav1 were incubated with oxLDL (100 μg ApoB/ml) for 8 or 14 hrs, then treated with sulfo‐NHS‐SS‐Biotin (1 mg/ml) and lysed. Biotinylated proteins were recovered as described under ‘Materials and methods’, and TRPC1 expression analysed by immunoblotting using an anti‐TRPC1 antibody. Total lysates were analysed with TRPC1 antibody to insure for equal loading of proteins. Results are representative of at least three independent experiments. (C), Translocation of TRPC1 into caveolae membranes upon oxLDL stimulation. SMC/cav1 were treated with oxLDL (100 μg ApoB/ml), after 8 hrs, cells were lysed in carbonate buffer and subjected to sucrose gradient sedimentation, as described under ‘Materials and methods’. A total of 50 μl of pooled fractions (4, 5 and 6 = L, light‐density fractions); 7, 8 and 9 = H1, high‐density fractions; 10, 11 and 12 = H2, high‐density fractions) were used for the detection of TRPC1 and caveolin‐1 by immunoblotting using anti‐TRPC1 and anti‐caveolin‐1 antibodies. The graph represents values of caveolin‐1 and TRPC1 band intensity by densitometry (a.u., arbitrary units). Results are representative of three independent experiments. (*P < 0.05, significantly different mean expression value from untreated conditions).

Article Snippet: The following antibodies were used: polyclonal anti‐TRPC1 (Alomone Labs, Jerusalem, Israel), polyclonal anti‐caveolin‐1 and monoclonal anti‐β‐actin (Upstate Biotechnology, Billerica, MA, USA).

Techniques: Translocation Assay, Expressing, Incubation, Western Blot, Sedimentation