trpc6  (Alomone Labs)

Journal: The Journal of Biological Chemistry

Article Title: Exploring the Structure of the Voltage-gated Na+ Channel by an Engineered Drug Access Pathway to the Receptor Site for Local Anesthetics *

doi: 10.1074/jbc.M113.541763

Figure Lengend Snippet: Structure of the external access pathway. The proposed molecular arrangement of crucial amino acids involved in access and binding of QX-222 is shown. A and B ), respectively, based

Article Snippet: To explore this possibility, we held the channels at a potential of −120 mV while applying QX-222 externally for 120 s. During this period, channels are expected to reside in the closed state.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Exploring the Structure of the Voltage-gated Na+ Channel by an Engineered Drug Access Pathway to the Receptor Site for Local Anesthetics *

doi: 10.1074/jbc.M113.541763

Figure Lengend Snippet: Recovery from block by intracellular QX-222. A 5-s-long 10-Hz pulse train was applied for repetitive openings of the channels to bind QX-222 to the inner vestibule. After the last pulse in the train, the cell was repolarized to −140 mV for various

Article Snippet: To explore this possibility, we held the channels at a potential of −120 mV while applying QX-222 externally for 120 s. During this period, channels are expected to reside in the closed state.

Techniques: Blocking Assay

Journal: The Journal of Biological Chemistry

Article Title: Exploring the Structure of the Voltage-gated Na+ Channel by an Engineered Drug Access Pathway to the Receptor Site for Local Anesthetics *

doi: 10.1074/jbc.M113.541763

Figure Lengend Snippet: The binding site for external QX-222 is in the internal vestibule. Modulation of the EAP by an additional mutation in the selectivity filter and by mutations of Trp-1531 is shown. The stimulation protocol and depiction are the same as in B. A , residue

Article Snippet: To explore this possibility, we held the channels at a potential of −120 mV while applying QX-222 externally for 120 s. During this period, channels are expected to reside in the closed state.

Techniques: Binding Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Exploring the Structure of the Voltage-gated Na+ Channel by an Engineered Drug Access Pathway to the Receptor Site for Local Anesthetics *

doi: 10.1074/jbc.M113.541763

Figure Lengend Snippet: Mutations at Site 1575 Open an EAP for QX-222

Article Snippet: To explore this possibility, we held the channels at a potential of −120 mV while applying QX-222 externally for 120 s. During this period, channels are expected to reside in the closed state.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Exploring the Structure of the Voltage-gated Na+ Channel by an Engineered Drug Access Pathway to the Receptor Site for Local Anesthetics *

doi: 10.1074/jbc.M113.541763

Figure Lengend Snippet: Effect of QX-222 on inactivation properties of channels carrying mutations at site 1531. A , in W1531A, 500 μ m QX-222 significantly shifted the steady-state inactivation curve to hyperpolarized potentials. V 1/2 was −74.6 ± 0.6 and

Article Snippet: To explore this possibility, we held the channels at a potential of −120 mV while applying QX-222 externally for 120 s. During this period, channels are expected to reside in the closed state.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Exploring the Structure of the Voltage-gated Na+ Channel by an Engineered Drug Access Pathway to the Receptor Site for Local Anesthetics *

doi: 10.1074/jbc.M113.541763

Figure Lengend Snippet: External block by QX-222 is favored by replacement of Ile-1575 with amino acids of small size or with hydrophilic properties. Sodium currents were evoked by depolarizing pulses at a frequency of 2 Hz (see “Experimental Procedures”). Connecting

Article Snippet: To explore this possibility, we held the channels at a potential of −120 mV while applying QX-222 externally for 120 s. During this period, channels are expected to reside in the closed state.

Techniques: Blocking Assay

Journal: The Journal of Biological Chemistry

Article Title: Exploring the Structure of the Voltage-gated Na+ Channel by an Engineered Drug Access Pathway to the Receptor Site for Local Anesthetics *

doi: 10.1074/jbc.M113.541763

Figure Lengend Snippet: Use-dependent block by external QX-222. A 2-Hz pulse train was applied for 30 s at a holding potential of −120 mV. Thereafter, external perfusion with QX-222 was started while cells were kept at the holding potential for 120 s. Then repetitive

Article Snippet: To explore this possibility, we held the channels at a potential of −120 mV while applying QX-222 externally for 120 s. During this period, channels are expected to reside in the closed state.

Techniques: Blocking Assay

Journal: Cancers

Article Title: The Role of the Anti-Aging Protein Klotho in IGF-1 Signaling and Reticular Calcium Leak: Impact on the Chemosensitivity of Dedifferentiated Liposarcomas

doi: 10.3390/cancers10110439

Figure Lengend Snippet: The intracellular localization of TRPC6 contributes to reticular Ca 2+ -leakage in KL -overexpressing DDLPS cells, but is not necessary to increase cell death. ( A – F ) Variations of relative cytosolic Ca 2+ concentration were monitored by fluorescence videomicroscopy in Fluo2-loaded cells. Each experiment was repeated three times and the average of more than 20 single-cell traces was analyzed. The effect of OAG (50 µM) addition was evaluated in ( A ) IB115-empty vector or ( B ) IB115-KL cells bathed in HBSS medium ± 2 mM Ca 2+ , and in ( C ) IB115-KL cells pretreated with TG (10 nM) at 200 s in a Ca 2+ -free HBSS medium. ( D ) IB115 empty-vector and IB115-KL cells were bathed in a Ca 2+ -free HBSS medium and stimulated at 200 s by the specific activator of TRPC6 Hyp9 (1 μM). ( E ) OAG (50 µM) was added at 200 s to Ca 2+ -free HBSS medium on IB115-KL cells control or pretreated with inhibitors of TRPC6 (U73343 and larixyl acetate both at 1 μM, 1 h). ( F ) Ca 2+ -leakage was estimated by the application of 10 nM TG at 200 s on IB115-empty vector or IB115-KL cells, which were pretreated or not with larixyl acetate (1 μM, 1 h), in a Ca 2+ -free HBSS medium. ( G ) The abundance of TRPC6 was analyzed in IB115-empty vector and IB115-KL cells by western blotting after 48 h of incubation with no drugs, 10 nM TG, or 100 nM gemcitabine. Actin was used as a loading control. To compare TRPC6 abundance between conditions, results were all normalized to control conditions (IB115-empty vector, no treatment). Results shown are representative of three independent experiments. ( H , I ) Cell death was measured with a TMRM-staining analyzed by flow cytometry after 72 h incubation of IB115 cell lines, which were pretreated during 1 h with indicated concentrations of larixyl acetate and then treated with ( H ) 20 nM and 10 nM TG for the IB115-empty vector and IB115-KL cells, respectively (in order to have nearly similar cell death rates), or ( I ) 100 nM gemcitabine. Histograms sum up ( H ) four and ( I ) two independent experiments. Data shown correspond to medians (IQR).

Article Snippet: Anti-TRPC6 (#ACC-120) was provided by Alomone Labs (Jerusalem, Israel).

Techniques: Concentration Assay, Fluorescence, Plasmid Preparation, Western Blot, Incubation, Staining, Flow Cytometry, Cytometry

Journal: Journal of Molecular Histology

Article Title: Localization of AQP5 during development of the mouse submandibular salivary gland

doi: 10.1007/s10735-010-9308-0

Figure Lengend Snippet: AQP5 expression pattern during postnatal development of the mouse submandibular gland (SMG). A At birth (postnatal day 0, P0), the pro-acinar cells ( arrow ) and the intercalated ducts ( ID ) are AQP5 positive. No AQP5 is detected in the intralobular duct ( IAD ) or interlobular duct ( IED ). B Pre-weaning (P5), both pro-acini ( arrow ) and intercalated ducts ( ID ) are AQP5 positive. No AQP5 is detected in the striated duct ( SD ). C Young adult females (P25): acini ( arrow ) and the proximal part of the intercalated duct ( ID ) are positive, while the granulated convoluted tubule ( GCT ) is negative. D Adult females (P60) show the same AQP5 pattern as in C . In addition, no AQP5 is detected in the transition from GCT to striated duct ( arrowhead ). E Adult males (P60): acini ( arrow ) and entire intercalated ducts ( ID ) are positive while the granulated convoluted tubule ( GCT ) is negative. F IgG negative control in young adult female (P25) tissue shows no unspecific staining in the acini ( arrow ), intercalated duct ( ID ), or in the granulated convoluted tubule ( GCT ). Granules in the granulated convoluted tubule are not seen using this method. A – F Scale bar 50 μm

Article Snippet: The sections were blocked for non-specific staining in 5% BSA (BSA, Sigma–Aldrich Corp., St. Louis, MO, USA) in PBS for 120 min before incubation overnight with anti-AQP5 (Alomone labs).

Techniques: Expressing, Negative Control, Staining

Journal: Journal of Molecular Histology

Article Title: Localization of AQP5 during development of the mouse submandibular salivary gland

doi: 10.1007/s10735-010-9308-0

Figure Lengend Snippet: Gold labeling of AQP5 in the SMG of adult animals (P60). A Overview of AQP5 gold staining in the basal membrane, membranes of intercellular canaliculi, as well as the lateral membrane ( LM ). CL canalicular lumen, BD basal digits (scale bar, 1 μm). B Gold labeling of AQP5 was detected in the apical membrane of acinar cells (scale bar, 1 μm). C Longitudinal section of an intercellular canaliculus showing gold labeling of AQP5 in the membrane (scale bar, 0.5 μm). D AQP5 was localized in the basal membrane in areas where digits could be seen, while no AQP5 was detectable in the basement membrane (scale bar 0.2 μm). Non-linear adjustments were applied to entire images

Article Snippet: The sections were blocked for non-specific staining in 5% BSA (BSA, Sigma–Aldrich Corp., St. Louis, MO, USA) in PBS for 120 min before incubation overnight with anti-AQP5 (Alomone labs).

Techniques: Labeling, Staining

Journal: Journal of Molecular Histology

Article Title: Localization of AQP5 during development of the mouse submandibular salivary gland

doi: 10.1007/s10735-010-9308-0

Figure Lengend Snippet: Confocal imaging of the adult SMG (P60). Aquaporin 5 (AQP5) demonstrates an apical, lateral, and basal localization in the SMG acini. A Localization of ZO-1 ( green ) near luminal membranes of acinar cells. B Localization of AQP5 ( red ) within the same cells as in A . C Merged image of A and B confirms the apical localization of AQP5. Arrow heads luminal membrane (scale bar 20 μm). D Localization of E-cadherin ( green ) within lateral membranes in both acinar ( arrow heads ) and GCT cells (*). E Localization of AQP5 ( red ) in the acinar cells. F Merged image of D and E demonstrates a lateral localization of AQP5 ( yellow / orange ), designated by arrowheads . An AQP5 and E-cadherin non-overlapping pattern of expression in the luminal canaliculi is evident ( arrow ). Non-overlapping patterns are also seen in the GCT cells (*) (scale bar, 50 μm). G Immunostaining of the basement membrane protein coll IV (blue). H Localization of AQP5 ( red ) in the acinar cells. I Merged image confirms a basal localization ( arrow head ) of AQP5 distinct from coll IV localization (scale bar 20 μm). J Localization of E-cadherin ( green ) within lateral membranes in acinar cells in a tissue immunostained with preabsorbed AQP5. K Preabsorption of the AQP5 antibody using its cognate peptide, showing complete elimination of the AQP5 staining pattern in the same cells as in J (scale bar 20 μm). A – I images are of male P60, J – K images are of female P60

Article Snippet: The sections were blocked for non-specific staining in 5% BSA (BSA, Sigma–Aldrich Corp., St. Louis, MO, USA) in PBS for 120 min before incubation overnight with anti-AQP5 (Alomone labs).

Techniques: Imaging, Expressing, Immunostaining, Staining

Journal: Journal of Molecular Histology

Article Title: Localization of AQP5 during development of the mouse submandibular salivary gland

doi: 10.1007/s10735-010-9308-0

Figure Lengend Snippet: AQP5 expression pattern during prenatal development of the mouse submandibular gland (SMG). A Pseudoglandular stage (~E14): no AQP5 staining is detectable in the terminal bud ( t ), epithelial stalk ( e ) or mesechyme ( m ). B Early canalicular stage (~E14-15): AQP5 negative cells in the terminal bud ( t ), presumptive duct ( pd ), and mesenchyme ( m ). C Late canalicular stage (~E15-E16): scattered positive pro-acinar cells are present ( arrow ). No AQP5 is found in the presumptive duct ( pd ) or mesenchyme ( m ). D Early terminal bud stage (~E16-E17): all pro-acinar cells are AQP5 positive ( arrow ). In the intralobular duct ( IAD ), cells proximal to the pro-acini show apical AQP5 staining ( arrowhead ). E and F Late terminal bud stage (~E17-18): a similar expression pattern is observed as in D . Additionally, the rest of the intralobular duct (IAD) is also positive, and the interlobular duct ( IED ) is negative. Scale bar ( A – E ) 50 μm and ( F ) 100 μm

Article Snippet: The sections were blocked for non-specific staining in 5% BSA (BSA, Sigma–Aldrich Corp., St. Louis, MO, USA) in PBS for 120 min before incubation overnight with anti-AQP5 (Alomone labs).

Techniques: Expressing, Staining

Journal: Vascular pharmacology

Article Title: GPR55 agonist lysophosphatidylinositol and lysophosphatidylcholine inhibit endothelial cell hyperpolarization via GPR-independent suppression of Na+-Ca2+ exchanger and endoplasmic reticulum Ca2+ refilling

doi: 10.1016/j.vph.2017.01.002

Figure Lengend Snippet: LPI suppresses the hyperpolarization to Ach, but not to SKA-31, in the endothelium of mice aorta. (A) Effect of 3 μM LPI on endothelial hyperpolarization to 2 μM Ach (n = 4). (B) Effect of 10 μM LPI on endothelial hyperpolarization to two consecutive administrations of 2 μM Ach (n = 3). The hyperpolarization to SKA-31 (10 μM) remained unaffected by LPI pre-exposure (n = 4). (C) Representative membrane potential recording from in situ mice aortic endothelium showing a failure of LPI (10 μM) to inhibit the hyperpolarization evoked by 10 μM SKA-31 (n = 3).

Article Snippet: LPC16:0 (1-palmitoyl-2-hydroxy- sn - glycero -3-phosphocholine) was purchased from Avanti Polar Lipids, LPI from Sigma Aldrich, paxilline and SKA-31 were purchased from Alomone Labs, KB-R7943 was purchased from TCI Chemicals.

Techniques: Mouse Assay, In Situ