Journal:

Article Title: Protein tyrosine kinase is expressed and regulates ROMK1 location in the cortical collecting duct

doi: 10.1152/ajprenal.00301.2003

Figure Lengend Snippet: Confocal images showing the colocalization of aquaporin-2 (AQP2; green) and c-Src (red) in the renal cortex (A) and OM (B) from rats on a normal-K diet. Bar = 10 µM or as indicated.

Article Snippet: Immunocytochemical staining The slides were washed with 1× PBS for 15 min and permeablized with 0.4% Triton dissolved in 1× PBS buffer containing 1% BSA and 0.1% lysine (pH 7.4) for 15 min. Kidney slices were blocked with 2% goat serum for 1 h at room temperature and then incubated with ROMK antibody (Alomone, Jerusalem, Israel), c-Src monoclonal antibody (Transduction Lab), aquaporin-2 (AQP2) antibody (Alomone), or Tamm-Horsfall glycoprotein (THP) antibody (ICN, Pharmaceutical, Aurora, OH) for 12 h at 4°C.

Techniques:

Journal:

Article Title: Protein tyrosine kinase is expressed and regulates ROMK1 location in the cortical collecting duct

doi: 10.1152/ajprenal.00301.2003

Figure Lengend Snippet: Magnified confocal image (×100) showing the double staining of AQP2 and c-Src in the renal cortex (A) and OM (B). Bar = 10 µM.

Article Snippet: Immunocytochemical staining The slides were washed with 1× PBS for 15 min and permeablized with 0.4% Triton dissolved in 1× PBS buffer containing 1% BSA and 0.1% lysine (pH 7.4) for 15 min. Kidney slices were blocked with 2% goat serum for 1 h at room temperature and then incubated with ROMK antibody (Alomone, Jerusalem, Israel), c-Src monoclonal antibody (Transduction Lab), aquaporin-2 (AQP2) antibody (Alomone), or Tamm-Horsfall glycoprotein (THP) antibody (ICN, Pharmaceutical, Aurora, OH) for 12 h at 4°C.

Techniques: Double Staining

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: Purinergic Signalling

Article Title: Purinergic receptors are part of a signalling system for proliferation and differentiation in distinct cell lineages in human anagen hair follicles

doi: 10.1007/s11302-008-9108-0

Figure Lengend Snippet: Expression of P2Y 1 and P2Y 2 receptors in human anagen hair follicles. a P2Y 1 receptors were found in the outer root sheath ( ORS ) and bulb of anagen hair follicles in longitudinal section but not in the inner root sheath ( IRS ). Scale bar = 40 μm. b In transverse section, P2Y 1 receptors were only seen in the outer root sheath ( ORS ). Scale bar = 40 μm. c P2Y 2 receptors were found in the cortex ( CTX ). Scale bar = 40 μm. d Transverse section of anagen hair follicle: P2Y 2 receptors were seen in the cortex ( CTX ) but not in the central medulla ( M ), inner ( IRS ) or outer root sheaths ( ORS ), or in the surrounding adventitial layer ( A ). Scale bar = 40 μm. e , f Controls: the immunoreaction was abolished after preabsorption of the e P2Y 1 and f P2Y 2 receptor antibodies with the corresponding peptides, confirming the specificity of the immunoreaction. Scale bars = 100 μm

Article Snippet: Polyclonal anti-P2Y 1 and P2Y 2 antibodies were obtained from Alomone Labs (Jerusalem, Israel) and corresponded to the third extracellular loop of the P2Y 1 (AA 242–258) and P2Y 2 receptor (AA 227–244).

Techniques: Expressing

Journal: Purinergic Signalling

Article Title: Purinergic receptors are part of a signalling system for proliferation and differentiation in distinct cell lineages in human anagen hair follicles

doi: 10.1007/s11302-008-9108-0

Figure Lengend Snippet: Double labelling of P2Y 1 and P2Y 2 receptors with markers for cellular proliferation, and double labelling of P2X 5 receptors with markers for keratinocyte differentiation in anagen hair follicles. a Double labelling of P2Y 1 receptors ( red ) with Ki-67, a nuclear marker for proliferating cells ( green ), to show that P2Y 1 receptors are found in proliferating basal cells in the outer root sheath ( ORS ) and bulb region of the hair follicle in longitudinal section. Scale bar = 50 μm. b Transverse section: double labelling of P2Y 1 receptors ( red ) with Ki-67, a nuclear marker ( green ), to show that P2Y 1 receptors are found in proliferating cells in the outer root sheath ( ORS ) of the hair follicle. Scale bar = 50 μm. c Longitudinal section of anagen hair follicle through the dermal papilla ( DP ): double labelling of P2Y 2 receptors ( red ) with proliferating cell nuclear antigen (PCNA), a marker for proliferating cells ( green ), showed that P2Y 2 receptors were found in the cortex ( CTX ) and at the edge of the medulla ( M ) but not in the central medulla. P2Y 2 receptors were not found in the matrix ( Ma ), where cells were positive for PCNA. Scale bar = 75 μm. d Longitudinal section of anagen hair follicle: P2Y 2 receptors were absent from the keratinised cuticle ( Cu ) of the hair shaft. PCNA was also found in cells of the outer root sheath ( ORS ). Scale bar = 75 μm. e Double labelling of P2X 5 receptors ( red-brown ) with involucrin, a marker for differentiating cells ( green ). Involucrin was expressed both in the inner root sheath ( IRS ), cortex ( CTX ) and in the outermost edge of the medulla ( M ). P2X 5 receptors were expressed in the inner ( IRS ) and outer root sheaths ( ORS ) and in the medulla ( M ) and matrix cells ( Ma ). There was yellow colocalisation with P2X 5 receptors in the inner root sheath and in cells at the outermost edge of the medulla ( arrow ). The cortex only stained positive for involucrin, not P2X 5 receptors. Scale bar = 50 μm. f Transverse section: double labelling of P2X 5 receptors ( red ) with involucrin ( green ). Involucrin was expressed in the inner root sheath ( IRS ) and cortex ( CTX ) and colocalised ( yellow ) with P2X 5 receptor staining in the inner root sheath ( IRS ). Scale bar = 50 μm

Article Snippet: Polyclonal anti-P2Y 1 and P2Y 2 antibodies were obtained from Alomone Labs (Jerusalem, Israel) and corresponded to the third extracellular loop of the P2Y 1 (AA 242–258) and P2Y 2 receptor (AA 227–244).

Techniques: Marker, Staining

Journal: American Journal of Human Genetics

Article Title: Identification of a Kir3.4 Mutation in Congenital Long QT Syndrome

doi: 10.1016/j.ajhg.2010.04.017

Figure Lengend Snippet: The Kir3.4-Gly387Arg Mutation Is Associated with Congenital Long QT Syndrome

Article Snippet: Proteins were transferred onto Hybond-P polyvinylidene difluoride transfer membranes (Amersham Biosciences, 0.45 μm), and membranes were blocked in 5% nonfat milk in TBST (0.1% Tween 20 in Tris-buffered saline solution) for 2 hr at room temperature and then incubated with primary antibody against Kir3.1 (1:1,000; Alomone Labs), Kir3.4 (1:1,000; Alomone Labs), calnexin (1:2,000; Nordic Biosite), or Na + /K+-ATPase α1 (1:500; Santa Cruz Biotechnology) overnight.

Techniques: Mutagenesis

Journal: American Journal of Human Genetics

Article Title: Identification of a Kir3.4 Mutation in Congenital Long QT Syndrome

doi: 10.1016/j.ajhg.2010.04.017

Figure Lengend Snippet: Clinical Characteristics of the Pedigree Members with Kir3.4-Gly387Arg or Sudden Death

Article Snippet: Proteins were transferred onto Hybond-P polyvinylidene difluoride transfer membranes (Amersham Biosciences, 0.45 μm), and membranes were blocked in 5% nonfat milk in TBST (0.1% Tween 20 in Tris-buffered saline solution) for 2 hr at room temperature and then incubated with primary antibody against Kir3.1 (1:1,000; Alomone Labs), Kir3.4 (1:1,000; Alomone Labs), calnexin (1:2,000; Nordic Biosite), or Na + /K+-ATPase α1 (1:500; Santa Cruz Biotechnology) overnight.

Techniques:

Journal: American Journal of Human Genetics

Article Title: Identification of a Kir3.4 Mutation in Congenital Long QT Syndrome

doi: 10.1016/j.ajhg.2010.04.017

Figure Lengend Snippet: Electrophysiological Analyses of Kir3.4-Gly387Arg

Article Snippet: Proteins were transferred onto Hybond-P polyvinylidene difluoride transfer membranes (Amersham Biosciences, 0.45 μm), and membranes were blocked in 5% nonfat milk in TBST (0.1% Tween 20 in Tris-buffered saline solution) for 2 hr at room temperature and then incubated with primary antibody against Kir3.1 (1:1,000; Alomone Labs), Kir3.4 (1:1,000; Alomone Labs), calnexin (1:2,000; Nordic Biosite), or Na + /K+-ATPase α1 (1:500; Santa Cruz Biotechnology) overnight.

Techniques:

Journal: American Journal of Human Genetics

Article Title: Identification of a Kir3.4 Mutation in Congenital Long QT Syndrome

doi: 10.1016/j.ajhg.2010.04.017

Figure Lengend Snippet: Cell Surface Expression of Kir3.4-Gly387Arg and Kir3.1

Article Snippet: Proteins were transferred onto Hybond-P polyvinylidene difluoride transfer membranes (Amersham Biosciences, 0.45 μm), and membranes were blocked in 5% nonfat milk in TBST (0.1% Tween 20 in Tris-buffered saline solution) for 2 hr at room temperature and then incubated with primary antibody against Kir3.1 (1:1,000; Alomone Labs), Kir3.4 (1:1,000; Alomone Labs), calnexin (1:2,000; Nordic Biosite), or Na + /K+-ATPase α1 (1:500; Santa Cruz Biotechnology) overnight.

Techniques: Expressing

Journal: American Journal of Human Genetics

Article Title: Identification of a Kir3.4 Mutation in Congenital Long QT Syndrome

doi: 10.1016/j.ajhg.2010.04.017

Figure Lengend Snippet: Human Atrial and Ventricular Expression of Kir3.1 and Kir3.4

Article Snippet: Proteins were transferred onto Hybond-P polyvinylidene difluoride transfer membranes (Amersham Biosciences, 0.45 μm), and membranes were blocked in 5% nonfat milk in TBST (0.1% Tween 20 in Tris-buffered saline solution) for 2 hr at room temperature and then incubated with primary antibody against Kir3.1 (1:1,000; Alomone Labs), Kir3.4 (1:1,000; Alomone Labs), calnexin (1:2,000; Nordic Biosite), or Na + /K+-ATPase α1 (1:500; Santa Cruz Biotechnology) overnight.

Techniques: Expressing

Journal:

Article Title: Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

doi: 10.1073/pnas.2436379100

Figure Lengend Snippet: Activation of ERK by anandamide in dormant blastocysts via CB1. (A) Localization of CB1 in dormant and activated blastocysts. The trophectoderm cell surface is decorated with CB1. The levels of CB1 are significantly lower in activated blastocysts than those in dormancy. (B) Rapid activation of ERK by anandamide (ANA) in blastocysts. Dormant blastocysts were cultured in vitro in the presence of 7 nM anandamide for the indicated times in minutes. Increased phosphorylation of ERK1/2(p-ERK1/2) and its translocation into nuclei were observed in dormant blastocyst trophectoderm cells within 5 min of their exposure to 7 nM anandamide, reaching a peak between 15 and 30 min. (C) Activation of ERK by anandamide is dose-dependent. Dormant blastocysts were cultured in vitro in the presence of 7 or 28 nM anandamide for 15 min. Anandamide at 7 nM activated ERK1/2 in dormant blastocyst trophectoderm cells, whereas it failed to do so at 28 nM. A CB1-selective antagonist SR141716A (SR1) at 7 nM or a MEK1/2 inhibitor U0126 at 1 μM inhibited the activation of ERK1/2 by 7 nM anandamide. (D) Total ERK remained unchanged. No changes in immunointensity for total ERK1/2 were observed in dormant blastocysts exposed to 7 nM anandamide or the vehicle. (E–G) Differential activation of ERK signaling by anandamide in CB mutant dormant blastocysts. CB1–/–, CB2–/–,or CB1–/– × CB2–/– dormant blastocysts were cultured in vitro in the presence of 7 nM anandamide. Activation of ERK1/2 in the presence of 7 nM anandamide for 15 min was abrogated by the CB1 antagonist SR141716A (SR1) in CB2–/– blastocyst, but not in CB1–/– or CB1–/– × CB2–/– blastocysts. Images shown depict TRITC-labeled antigens in red, Hoechst-labeled nuclei in blue, and the merge in pink. (Scale bars, 20 μm.)

Article Snippet: Rabbit polyclonal antibodies specific to CB1 (1 μg/ml, custom made), or α 1A , α 1B , and α1C (1 μg/ml, Alomone Labs, Jerusalem), or total and phospho-ERK 1 and 2 (ERK1/2) (0.5 μg/ml, New England Biolabs) were used.

Techniques: Activation Assay, Cell Culture, In Vitro, Translocation Assay, Mutagenesis, Labeling

Journal:

Article Title: Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

doi: 10.1073/pnas.2436379100

Figure Lengend Snippet: Activation of ERK by anandamide in normal day-4 blastocysts via CB1. Day-4 blastocysts were cultured in vitro in the presence of 7 or 28 nM anandamide (ANA) for 15 min. Activation of ERK1/2 at lower (7 nM) but not higher (28 nM) anandamide concentration was observed. A CB1-selective antagonist SR141716A (SR1) inhibited the accumulation of phospho-ERK1/2 (p-ERK1/2) by 7 nM anandamide. Images depict TRITC-labeled antigen in red, Hoechst-labeled nuclei in blue, and the merge in pink. (Scale bar, 20 μm.) Tr, trophectoderm; ICM, inner cell mass.

Article Snippet: Rabbit polyclonal antibodies specific to CB1 (1 μg/ml, custom made), or α 1A , α 1B , and α1C (1 μg/ml, Alomone Labs, Jerusalem), or total and phospho-ERK 1 and 2 (ERK1/2) (0.5 μg/ml, New England Biolabs) were used.

Techniques: Activation Assay, Cell Culture, In Vitro, Concentration Assay, Labeling

Journal:

Article Title: Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

doi: 10.1073/pnas.2436379100

Figure Lengend Snippet: Cannabinoid agonist CP55,940 induces activation of ERK in differentiating TS cells via CB1. (A) CB1 is expressed in TS cells. This cell line is stably transfected with the GFP gene. Images depict GFP in green, CB1 in red (TS cell surfaces), and the merge in yellow. (Scale bar, 50 μm.) (B and C) Activation of ERK in TS cells by CP55,940 (CP). TS cells were plated and expanded for 48 h. The cells were serum-starved for 5 h then exposed to different concentrations of CP for 15 min or 7 nM CP for the indicated times or to CP at 7 nM in the presence or absence of a MEK1/2 inhibitor (U0126), CB1-selective antagonist SR141716A (SR1), or CB2-selective antagonist SR144528 (SR2) for 5 min. Phosphorylation of ERK1 in differentiating TS cells was rapidly induced by 7 nM CP. U0126 or SR1, but not SR2, inhibited this activation. Quantitative analysis of ERK activation in C is expressed as percentage relative to the maximum band intensity.

Article Snippet: Rabbit polyclonal antibodies specific to CB1 (1 μg/ml, custom made), or α 1A , α 1B , and α1C (1 μg/ml, Alomone Labs, Jerusalem), or total and phospho-ERK 1 and 2 (ERK1/2) (0.5 μg/ml, New England Biolabs) were used.

Techniques: Activation Assay, Stable Transfection, Transfection

Journal:

Article Title: Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

doi: 10.1073/pnas.2436379100

Figure Lengend Snippet: Inhibition of depolarization-induced Ca2+ influx by 28 nM anandamide in dormant blastocysts. (A) Distribution of Ca2+ channel α subunits, α1B (N-type) and α1C (L-type), in dormant blastocysts. Both inner cell mass (ICM) and trophectoderm cell (Tr) are decorated with α1B and α1C subunits shown in red, Hoechst-labeled nuclei in blue, and the merge in pink. (B) Inhibition of depolarization-induced Ca2+ influx by anandamide (ANA). Ca2+ mobilization in blastocysts was visualized with Fluo-4 acetoxymethyl ester. Depolarization-induced Ca2+ influx in dormant blastocysts after exposure to 60 mM KCl was dramatically inhibited by 28 nM anandamide but not by 7 nM. The CB1-selective antagonist SR141716A (SR1) at equimolar concentration reversed this inhibition, but the CB2-selective antagonist SR144528 (SR2) was ineffective. The relative level of intracellular Ca2+ is indicated by the fluorescent intensity, which is displayed in pseudocolor according to the color bar by using lsmib. (Scale bar, 20 μm.)

Article Snippet: Rabbit polyclonal antibodies specific to CB1 (1 μg/ml, custom made), or α 1A , α 1B , and α1C (1 μg/ml, Alomone Labs, Jerusalem), or total and phospho-ERK 1 and 2 (ERK1/2) (0.5 μg/ml, New England Biolabs) were used.

Techniques: Inhibition, Labeling, Concentration Assay

Journal:

Article Title: Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

doi: 10.1073/pnas.2436379100

Figure Lengend Snippet: Anandamide at 7 nM confers blastocyst competency to implantation via CB1

Article Snippet: Rabbit polyclonal antibodies specific to CB1 (1 μg/ml, custom made), or α 1A , α 1B , and α1C (1 μg/ml, Alomone Labs, Jerusalem), or total and phospho-ERK 1 and 2 (ERK1/2) (0.5 μg/ml, New England Biolabs) were used.

Techniques: