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Santa Cruz Biotechnology anti na k atpase beta 1 subunit
Anti Na K Atpase Beta 1 Subunit, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 94 stars, based on 47 article reviews

anti na k atpase beta 1 subunit - by Bioz Stars, 2026-03

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Both Helicobacter pylori infection and reduced Na,K-ATPase activity impair intracellular junctions in cultured cells ( A – C ) and in gerbil mucosa ( D ). HGE-20 cells were grown to confluency in Transwell™ inserts, and the transepithelial electrical resistance (TEER) was measured in the presence of H. pylori ( A ) or ouabain ( B ). The TEER is expressed as a percentage of the initial value. Initial values of the TEER in HGE-20 cells ranged from 1000 to 1500 Ω×cm 2 . Paracellular permeability of confluent HGE-20 cell layers was measured in an Ussing chamber in the presence or absence of ouabain, results are expressed as the rate of FD4 flux through the cell layer ( C ). Gerbils were euthanized and stomach tissue incubated in an Ussing chamber in the presence of ouabain, and the TEER was measured at the indicated times. Results are expressed as percent of initial value ( D ). Initial values of the TEER in gerbil gastric mucosa ranged from 75 to 100 Ω×cm 2 , consistent with prior published results . For all panels, n = 3, * p < 0.05 ( t -test), mean ± SD.

Journal: Biomolecules

Article Title: Helicobacter pylori -Induced Decrease in Membrane Expression of Na,K-ATPase Leads to Gastric Injury

doi: 10.3390/biom14070772

Figure Lengend Snippet: Both Helicobacter pylori infection and reduced Na,K-ATPase activity impair intracellular junctions in cultured cells ( A – C ) and in gerbil mucosa ( D ). HGE-20 cells were grown to confluency in Transwell™ inserts, and the transepithelial electrical resistance (TEER) was measured in the presence of H. pylori ( A ) or ouabain ( B ). The TEER is expressed as a percentage of the initial value. Initial values of the TEER in HGE-20 cells ranged from 1000 to 1500 Ω×cm 2 . Paracellular permeability of confluent HGE-20 cell layers was measured in an Ussing chamber in the presence or absence of ouabain, results are expressed as the rate of FD4 flux through the cell layer ( C ). Gerbils were euthanized and stomach tissue incubated in an Ussing chamber in the presence of ouabain, and the TEER was measured at the indicated times. Results are expressed as percent of initial value ( D ). Initial values of the TEER in gerbil gastric mucosa ranged from 75 to 100 Ω×cm 2 , consistent with prior published results . For all panels, n = 3, * p < 0.05 ( t -test), mean ± SD.

Article Snippet: Primary antibodies against Na,K-ATPase α 1 subunits (1:1000; mouse, Clone:C464.6, EMD, Millipore, Temecula, CA, USA) and Na-K-ATPase β1-subunits (1:1000; mouse, clone M17-P5-F11, Affinity Bioreagents, Golden, CO, USA) were used to detect Na,K-ATPase.

Techniques: Infection, Activity Assay, Cell Culture, Permeability, Incubation

Human gastric organoids as a model system to study the effect of Helicobacter pylori on gastric mucosa. Human gastric antrum was obtained from de-identified sleeve gastrectomy specimens. The location of the harvested sample is indicated by the circle ( A ). Human gastric organoids were cultivated from the fresh tissue samples and plated in Matrigel with appropriate growth factors, leading to the formation of 3D organoids ( B ). Basolateral expression of Na,K-ATPase was confirmed in gastric organoids by immunofluorescence of the α 1 subunit, indicated by the red color, and F-actin was used as a counterstain, indicated by the green color ( C ). Three-dimensional organoids were converted to 2D using Matrigel-coated Transwell™ inserts and were grown into a confluent monolayer. Organoids were incubated with the vehicle or H. pylori ( D , E ), and the decrease in Na,K-ATPase subunits in the presence of H. pylori was confirmed in this model system by immunofluorescence ( D ) or Western blot ( E ). Quantification is shown in the right panels. Mean ± SD, n = 3, * p < 0.05, t -test.

Journal: Biomolecules

Article Title: Helicobacter pylori -Induced Decrease in Membrane Expression of Na,K-ATPase Leads to Gastric Injury

doi: 10.3390/biom14070772

Figure Lengend Snippet: Human gastric organoids as a model system to study the effect of Helicobacter pylori on gastric mucosa. Human gastric antrum was obtained from de-identified sleeve gastrectomy specimens. The location of the harvested sample is indicated by the circle ( A ). Human gastric organoids were cultivated from the fresh tissue samples and plated in Matrigel with appropriate growth factors, leading to the formation of 3D organoids ( B ). Basolateral expression of Na,K-ATPase was confirmed in gastric organoids by immunofluorescence of the α 1 subunit, indicated by the red color, and F-actin was used as a counterstain, indicated by the green color ( C ). Three-dimensional organoids were converted to 2D using Matrigel-coated Transwell™ inserts and were grown into a confluent monolayer. Organoids were incubated with the vehicle or H. pylori ( D , E ), and the decrease in Na,K-ATPase subunits in the presence of H. pylori was confirmed in this model system by immunofluorescence ( D ) or Western blot ( E ). Quantification is shown in the right panels. Mean ± SD, n = 3, * p < 0.05, t -test.

Techniques: Expressing, Immunofluorescence, Incubation, Western Blot

Helicobacter pylori infection, reduced amount of Na,K-ATPase, and reduced Na,K-ATPase activity similarly decrease the TEER in human gastric organoids. ( A , B ) The TEER of 2D human gastric organoids grown on Transwell™ inserts and measured in an Endohm ® chamber was decreased after exposure to Helicobacter pylori ( A ) and in the presence of ouabain compared with the vehicle ( B ). Western blot showing the silencing of the Na,K-ATPase α 1 subunit ( C ). The TEER of 2D human gastric organoids grown on Transwell™ inserts and measured in an Endohm ® chamber was decreased after silencing the Na,K-ATPase α 1 subunit ( D ). Initial values of the TEER in 2D gastric organoids ranged from 800 to 1300 Ω×cm 2 . Mean ± SD, significant differences from control, t -test, n = 3, * p < 0.05 ( A , C , D ) and n = 5, ** p < 0.01 ( B ).

Journal: Biomolecules

Article Title: Helicobacter pylori -Induced Decrease in Membrane Expression of Na,K-ATPase Leads to Gastric Injury

doi: 10.3390/biom14070772

Figure Lengend Snippet: Helicobacter pylori infection, reduced amount of Na,K-ATPase, and reduced Na,K-ATPase activity similarly decrease the TEER in human gastric organoids. ( A , B ) The TEER of 2D human gastric organoids grown on Transwell™ inserts and measured in an Endohm ® chamber was decreased after exposure to Helicobacter pylori ( A ) and in the presence of ouabain compared with the vehicle ( B ). Western blot showing the silencing of the Na,K-ATPase α 1 subunit ( C ). The TEER of 2D human gastric organoids grown on Transwell™ inserts and measured in an Endohm ® chamber was decreased after silencing the Na,K-ATPase α 1 subunit ( D ). Initial values of the TEER in 2D gastric organoids ranged from 800 to 1300 Ω×cm 2 . Mean ± SD, significant differences from control, t -test, n = 3, * p < 0.05 ( A , C , D ) and n = 5, ** p < 0.01 ( B ).

Techniques: Infection, Activity Assay, Western Blot, Control

Reduced Na,K-ATPase activity and Helicobacter infection similarly impair adherens junctions in human gastric organoids. Immunofluorescence of 2D gastric organoids using E-cadherin antibodies showed a decreased E-cadherin signal at the cell junctions in the presence of H. pylori ( A ) or ouabain ( D ) and an increased intracellular accumulation of E-cadherin ( B , E ). E-cadherin clusters were decreased in size in the presence of H. pylori ( C ) or ouabain ( F ), suggesting injury to junctions. Mean ± SD, significant differences from “no Hp” or “no ouabain”, t -test. n = 5, ** p < 0.01, *** p < 0.001 ( B , F ).

Journal: Biomolecules

Article Title: Helicobacter pylori -Induced Decrease in Membrane Expression of Na,K-ATPase Leads to Gastric Injury

doi: 10.3390/biom14070772

Figure Lengend Snippet: Reduced Na,K-ATPase activity and Helicobacter infection similarly impair adherens junctions in human gastric organoids. Immunofluorescence of 2D gastric organoids using E-cadherin antibodies showed a decreased E-cadherin signal at the cell junctions in the presence of H. pylori ( A ) or ouabain ( D ) and an increased intracellular accumulation of E-cadherin ( B , E ). E-cadherin clusters were decreased in size in the presence of H. pylori ( C ) or ouabain ( F ), suggesting injury to junctions. Mean ± SD, significant differences from “no Hp” or “no ouabain”, t -test. n = 5, ** p < 0.01, *** p < 0.001 ( B , F ).

Techniques: Activity Assay, Infection, Immunofluorescence

a RT-PCR analyses of injured/non-regenerated (INR) and injured/regenerated (IR) RGCs ( N = 3 in each group). Results show Na + /K + -ATPase-α (i) and β (ii) mRNA expression levels relative to 18 S RNA expression levels (y-axis) in INR and IR RGCs ( x axis). b Immunoblot analyses of INR and IR RGCs ( N = 3 in each group). INR and IR RGCs were lysed and immunoblots of the cell lysates were probed using monoclonal antibodies raised against the α-subunit (i; 100-kDa) and β-subunit (ii; 46-, 60-kDa) of Na + /K + -ATPase, and anti-β-actin (42-kDa, panel below each blot). The protein expression was normalized against β-actin. Quantification of the immunoblots for the α-subunit (iii) and β-subunit (iv) of Na + /K + -ATPase. Full blot images are provided in Supplementary Data . c The number of regenerated axons ( y axis) amongst the control ( N = 7; 895 ± 163) and digoxin-treated ( N = 8; 605 ± 190) animals ( x axis) are depicted. A significant reduction in the number of regenerating cells was found following digoxin-mediated inhibition of the Na + /K + -ATPase. Values shown are mean ± SEM and all asterisks indicate a P value <0.05 from a Student’s t test. d , e The number of regenerated RGC axons in α1 ( c ; N = 6) and α2 ( d ; N = 10) heterozygous knockout mice ( y axis) was compared to those in α1 ( c ; N = 7) and α2 ( d ; N = 10) wild-type control mice ( x axis). Values shown are mean ± SEM and all asterisks indicate a P value <0.05 from a Student’s t test. Figure and Video 1 show a representative image (×20) of retina flat mount with the fluorescent (Fluorogold-positive) RGCs that regenerated axons to the end of the graft. The fluorescent cells are counted under fluorescent microscopy to generate the data in c – e .

Journal: Communications Biology

Article Title: Purified regenerating retinal neurons reveal regulatory role of DNA methylation-mediated Na+/K+-ATPase in murine axon regeneration

doi: 10.1038/s42003-023-04463-4

Figure Lengend Snippet: a RT-PCR analyses of injured/non-regenerated (INR) and injured/regenerated (IR) RGCs ( N = 3 in each group). Results show Na + /K + -ATPase-α (i) and β (ii) mRNA expression levels relative to 18 S RNA expression levels (y-axis) in INR and IR RGCs ( x axis). b Immunoblot analyses of INR and IR RGCs ( N = 3 in each group). INR and IR RGCs were lysed and immunoblots of the cell lysates were probed using monoclonal antibodies raised against the α-subunit (i; 100-kDa) and β-subunit (ii; 46-, 60-kDa) of Na + /K + -ATPase, and anti-β-actin (42-kDa, panel below each blot). The protein expression was normalized against β-actin. Quantification of the immunoblots for the α-subunit (iii) and β-subunit (iv) of Na + /K + -ATPase. Full blot images are provided in Supplementary Data . c The number of regenerated axons ( y axis) amongst the control ( N = 7; 895 ± 163) and digoxin-treated ( N = 8; 605 ± 190) animals ( x axis) are depicted. A significant reduction in the number of regenerating cells was found following digoxin-mediated inhibition of the Na + /K + -ATPase. Values shown are mean ± SEM and all asterisks indicate a P value <0.05 from a Student’s t test. d , e The number of regenerated RGC axons in α1 ( c ; N = 6) and α2 ( d ; N = 10) heterozygous knockout mice ( y axis) was compared to those in α1 ( c ; N = 7) and α2 ( d ; N = 10) wild-type control mice ( x axis). Values shown are mean ± SEM and all asterisks indicate a P value <0.05 from a Student’s t test. Figure and Video 1 show a representative image (×20) of retina flat mount with the fluorescent (Fluorogold-positive) RGCs that regenerated axons to the end of the graft. The fluorescent cells are counted under fluorescent microscopy to generate the data in c – e .

Article Snippet: The membrane was then incubated overnight at 4 °C with a mouse monoclonal antibody raised against the Na + /K + -ATPase α-subunit (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or with a mouse monoclonal antibody raised against the Na + /K + -ATPase β-subunit (1:500; Santa Cruz).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, RNA Expression, Western Blot, Bioprocessing, Control, Inhibition, Knock-Out, Microscopy

A . Agarose gel showing the breakdown of Yabro to lower molecular weight fragments (MW<500kD) by sonication for 1.5 min. The gel is stained with stain all. 1. HA-High ladder. 2. HA-Low ladder. 3. HMW-HA = Yabro. 4. LMW-HA. B . Instillation of 50μl of 150μg/ml LMW-HA to mice inhibited AFC starting at 4hrs and lasted at least 24hrs. Instillation of 50μl NPS2143 (1μM) at 6hrs post LMW-HA or 50 μl of HMW-HA (150μg/ml) reversed AFC to control values. C . Lung W/D ratio of 3 mice instilled with 50μl saline, 3 instilled with 50μl of LMW-HA at 150μg/ml, and 3 instilled with 50μl of NPS2143 at 1μM 6hrs post-LMW-HA. LMW-HA increased lung D/W 20%, while NPS2143 reduced it by 16%. D, E and F . Representative μOCT images of trachea from mice instilled with saline (D), 50μl of 150μg/ml HMW-HA (E), and 50μl of 1μM NPS2143 (F) at 6hrs post LMW-HA instillation. The red bar represents the thickness of the ELF. G . ELF increased threefold, from 14.9±2.1μm to 45.8±6.5μm, 24hrs post LMW-HA instillation. Inhibiting CaSR 6hrs post LMW-HA instillation with 1μM NPS2143 reduced ELF thickness by almost half, from 45.8±6.5μm to 24.8±4.8μm. Data are Means ± SE, n= 3-6. Significance was determined by 1-way ANOVA and post hoc Tukey test for multiple comparisons.

Journal: bioRxiv

Article Title: Low Molecular Weight Hyaluronan Inhibits Lung Epithelial Ion Channels by Activating the Calcium-Sensing Receptor

doi: 10.1101/2022.09.07.506946

Figure Lengend Snippet: A . Agarose gel showing the breakdown of Yabro to lower molecular weight fragments (MW<500kD) by sonication for 1.5 min. The gel is stained with stain all. 1. HA-High ladder. 2. HA-Low ladder. 3. HMW-HA = Yabro. 4. LMW-HA. B . Instillation of 50μl of 150μg/ml LMW-HA to mice inhibited AFC starting at 4hrs and lasted at least 24hrs. Instillation of 50μl NPS2143 (1μM) at 6hrs post LMW-HA or 50 μl of HMW-HA (150μg/ml) reversed AFC to control values. C . Lung W/D ratio of 3 mice instilled with 50μl saline, 3 instilled with 50μl of LMW-HA at 150μg/ml, and 3 instilled with 50μl of NPS2143 at 1μM 6hrs post-LMW-HA. LMW-HA increased lung D/W 20%, while NPS2143 reduced it by 16%. D, E and F . Representative μOCT images of trachea from mice instilled with saline (D), 50μl of 150μg/ml HMW-HA (E), and 50μl of 1μM NPS2143 (F) at 6hrs post LMW-HA instillation. The red bar represents the thickness of the ELF. G . ELF increased threefold, from 14.9±2.1μm to 45.8±6.5μm, 24hrs post LMW-HA instillation. Inhibiting CaSR 6hrs post LMW-HA instillation with 1μM NPS2143 reduced ELF thickness by almost half, from 45.8±6.5μm to 24.8±4.8μm. Data are Means ± SE, n= 3-6. Significance was determined by 1-way ANOVA and post hoc Tukey test for multiple comparisons.

Article Snippet: Commercially available antibodies to α-ENaC (H59, Santa Cruz), β-ENaC (sc-25354, Santa Cruz), and γ-ENaC (ab-3468, Abcam), CFTR protein (ab2478, Abcam) and Na,K-ATPase α-subunit (ab76020, Abcam) and β1-subunit (sc21713, Santa Cruz) were used to assess the expression of each ion channel.

Techniques: Agarose Gel Electrophoresis, Molecular Weight, Sonication, Staining, Control, Saline

ENaC activity in mouse ATII cells in-situ showing two conductances 4pS and 18pS before and during exposure to LMW-HA (Panels 4A, 4B, 4D, and 4E), the arrows indicate the open (o) and closed (c) states of the channels. ENaC activity was restored when CaSR was inhibited with 1μM NPS-2143 (Panels 4C, and 4F). Panels 4G and 4H summarize the open probability of ENaC in AT2 cells in control, 2-, 4-, and 24hrs post LMW-HA instillation to mice. LMW-HA inhibitory effect on ENaC at 4hrs post LMW-HA instillation and was reversed only when CaSR was inhibited with 1μM NPS2143 instilled 6hrs post LMW-HA. Data are means ± SE, n = 10 slices prepared from three mice in each group. Significance was determined by 1-way ANOVA and post hoc Tukey test for multiple comparisons.

Journal: bioRxiv

Article Title: Low Molecular Weight Hyaluronan Inhibits Lung Epithelial Ion Channels by Activating the Calcium-Sensing Receptor

doi: 10.1101/2022.09.07.506946

Figure Lengend Snippet: ENaC activity in mouse ATII cells in-situ showing two conductances 4pS and 18pS before and during exposure to LMW-HA (Panels 4A, 4B, 4D, and 4E), the arrows indicate the open (o) and closed (c) states of the channels. ENaC activity was restored when CaSR was inhibited with 1μM NPS-2143 (Panels 4C, and 4F). Panels 4G and 4H summarize the open probability of ENaC in AT2 cells in control, 2-, 4-, and 24hrs post LMW-HA instillation to mice. LMW-HA inhibitory effect on ENaC at 4hrs post LMW-HA instillation and was reversed only when CaSR was inhibited with 1μM NPS2143 instilled 6hrs post LMW-HA. Data are means ± SE, n = 10 slices prepared from three mice in each group. Significance was determined by 1-way ANOVA and post hoc Tukey test for multiple comparisons.

Techniques: Activity Assay, In Situ, Control

A . SCC recorded from MTEC monolayer. The basal current of 40μm/cm 2 is mostly amiloride sensitive carried by Na + ion entering the cell through ENaC. Forskolin (10uM) activated current and inhibited with CFTR-inhib-172 is a Cl − current through CFTR. The Na,K-ATPase current visible when apical membranes are permeabilized with amphotericin-B and sensitive to ouabain. B . Apical application of 150μg/ml LMW-HA to MTEC monolayers inhibited the amiloride sensitive current as early as 4hrs without affecting the CFTR- ihib.172 or ouabain sensitive currents. C . 24hrs post incubation with 150μg/ml LMW-HA all currents were inhibited. D . Summary of SSC at 4hrs and 24hrs post MTEC monolayers incubation with 150μg/ml LMW-HA. ENaC is inhibited as early as 4hrs. At 24hrs, in addition to ENaC, CFTR and Na,K-ATPase, are also inhibited. E . LMW-HA inhibition of SCC at 24hrs was reversed by apical application of 150μg/ml HMW-HA at 6hrs post 150μg/ml LMW-HA application to MTE monolayers. G . CaSR inhibition with 1μm NPS2143 at 6hrs post 150μg/ml LMW-HA application to MTE monolayers reversed LMW-HA effect on SCC. Data are means ± SE, n= 13-15, p<0.001. Significance was determined by 1-way ANOVA and post hoc Tukey test for multiple comparisons.

Journal: bioRxiv

Article Title: Low Molecular Weight Hyaluronan Inhibits Lung Epithelial Ion Channels by Activating the Calcium-Sensing Receptor

doi: 10.1101/2022.09.07.506946

Figure Lengend Snippet: A . SCC recorded from MTEC monolayer. The basal current of 40μm/cm 2 is mostly amiloride sensitive carried by Na + ion entering the cell through ENaC. Forskolin (10uM) activated current and inhibited with CFTR-inhib-172 is a Cl − current through CFTR. The Na,K-ATPase current visible when apical membranes are permeabilized with amphotericin-B and sensitive to ouabain. B . Apical application of 150μg/ml LMW-HA to MTEC monolayers inhibited the amiloride sensitive current as early as 4hrs without affecting the CFTR- ihib.172 or ouabain sensitive currents. C . 24hrs post incubation with 150μg/ml LMW-HA all currents were inhibited. D . Summary of SSC at 4hrs and 24hrs post MTEC monolayers incubation with 150μg/ml LMW-HA. ENaC is inhibited as early as 4hrs. At 24hrs, in addition to ENaC, CFTR and Na,K-ATPase, are also inhibited. E . LMW-HA inhibition of SCC at 24hrs was reversed by apical application of 150μg/ml HMW-HA at 6hrs post 150μg/ml LMW-HA application to MTE monolayers. G . CaSR inhibition with 1μm NPS2143 at 6hrs post 150μg/ml LMW-HA application to MTE monolayers reversed LMW-HA effect on SCC. Data are means ± SE, n= 13-15, p<0.001. Significance was determined by 1-way ANOVA and post hoc Tukey test for multiple comparisons.

Techniques: Inhibition, Incubation

A . Total proteins for α-, β-, and γ-ENaC, CFTR, α-β-Na,K-ATPase subunits and CaSR. Control (lanes 1-2). 150μg/ml LMW-HA 4hrs (lanes 3-4). LMW-HA 24hrs (lanes 5-6), LMW-HA + 1μM NPS-2143 (lanes 7-8), and LMW-HA + HMW-HA (lanes 9-10). LMW-HA reduced ENaC subunits, CFTR, Na,K-ATPase subunits expression as early as 4hrs. However, it stimulated the expression of CaSR protein expression. 1μ M NPS-2143 and 150μg/ml HMW-HA restored ENaC subunits, CFTR, Na,K-ATPase subunits, and CaSR expression to their control levels. B-H . Quantification of western blot data in A for ENaC subunits, CFTR, Na,K-ATPase subunits and CaSR. Combined results of different 5 blots from 5 different MTEC cultures. Data are Means ± SE, n=5. Significance was determined by 1-way ANOVA and post hoc Tukey test for multiple comparisons.

Journal: bioRxiv

Article Title: Low Molecular Weight Hyaluronan Inhibits Lung Epithelial Ion Channels by Activating the Calcium-Sensing Receptor

doi: 10.1101/2022.09.07.506946

Figure Lengend Snippet: A . Total proteins for α-, β-, and γ-ENaC, CFTR, α-β-Na,K-ATPase subunits and CaSR. Control (lanes 1-2). 150μg/ml LMW-HA 4hrs (lanes 3-4). LMW-HA 24hrs (lanes 5-6), LMW-HA + 1μM NPS-2143 (lanes 7-8), and LMW-HA + HMW-HA (lanes 9-10). LMW-HA reduced ENaC subunits, CFTR, Na,K-ATPase subunits expression as early as 4hrs. However, it stimulated the expression of CaSR protein expression. 1μ M NPS-2143 and 150μg/ml HMW-HA restored ENaC subunits, CFTR, Na,K-ATPase subunits, and CaSR expression to their control levels. B-H . Quantification of western blot data in A for ENaC subunits, CFTR, Na,K-ATPase subunits and CaSR. Combined results of different 5 blots from 5 different MTEC cultures. Data are Means ± SE, n=5. Significance was determined by 1-way ANOVA and post hoc Tukey test for multiple comparisons.

Techniques: Control, Expressing, Western Blot

Application of PLC, PKC, and ERK inhibitors to MTEC monolayers 4hrs prior to cells exposure to 150ug/ml LMW-HA. A . 4hrs post LMW-HA application ENaC current was protected from LMW-HA when PLC or PKC were inhibited but not ERK. B, C, and D . 24hrs post LMA-HA apical application to MTEC monolayers, all currents were protected by inhibition of either PLC, PKC, or ERK. Data are Means ± SE, n=10. Significance was determined by 1-way ANOVA and post hoc Tukey test for multiple comparisons.

Journal: bioRxiv

Article Title: Low Molecular Weight Hyaluronan Inhibits Lung Epithelial Ion Channels by Activating the Calcium-Sensing Receptor

doi: 10.1101/2022.09.07.506946

Figure Lengend Snippet: Application of PLC, PKC, and ERK inhibitors to MTEC monolayers 4hrs prior to cells exposure to 150ug/ml LMW-HA. A . 4hrs post LMW-HA application ENaC current was protected from LMW-HA when PLC or PKC were inhibited but not ERK. B, C, and D . 24hrs post LMA-HA apical application to MTEC monolayers, all currents were protected by inhibition of either PLC, PKC, or ERK. Data are Means ± SE, n=10. Significance was determined by 1-way ANOVA and post hoc Tukey test for multiple comparisons.

Techniques: Inhibition

Nasal potential different was measured in control and chlorine exposed mice (400ppm for 30min). Cl 2 inhalation reduced both the amiloride sensitive ( A ) and the Forskolin activated GlyH-101 inhibited ( B ) components by 64% and 69%, respectively, at 24hrs. Mice instillation with 50μl of 150μg/ml HMW-HA or 50μl of 1μM NPS-2143 at 6hrs post exposure reversed the effect of Cl 2 on NPD to 90%, and 80% of control values, respectively. Data are means ± SE, n= 13-15, p<0.001. Significance was determined by 1-way ANOVA and post hoc Tukey test for multiple comparisons.

Journal: bioRxiv

Article Title: Low Molecular Weight Hyaluronan Inhibits Lung Epithelial Ion Channels by Activating the Calcium-Sensing Receptor

doi: 10.1101/2022.09.07.506946

Figure Lengend Snippet: Nasal potential different was measured in control and chlorine exposed mice (400ppm for 30min). Cl 2 inhalation reduced both the amiloride sensitive ( A ) and the Forskolin activated GlyH-101 inhibited ( B ) components by 64% and 69%, respectively, at 24hrs. Mice instillation with 50μl of 150μg/ml HMW-HA or 50μl of 1μM NPS-2143 at 6hrs post exposure reversed the effect of Cl 2 on NPD to 90%, and 80% of control values, respectively. Data are means ± SE, n= 13-15, p<0.001. Significance was determined by 1-way ANOVA and post hoc Tukey test for multiple comparisons.

Techniques: Control

ENaC activity in human AT2 cells in-situ in precision cut lung slices from human lung tissue of a patient (more details are needed). A . Control recording from an ATII cells bathed in Ringer’s solution. Two conductance are visible, a 4pS and an 18 pS, the arrows indicate the open (o) and closed (c) states of the channels. B . Exposure of human lung slices to 150μg/ml LMW-HA for 4hrs reduced channels activities of conductances. C . incubation 1μM NPS-2143 abrogated LMW-HA effect on 18pS conductance but not on 4pS. D and E depict the summary data of both channels activities in control, 4hrs post exposure to 150μg/ml LMW-HA, and 4hrs post incubation with 150μg/ml LMW-HA and 1μM NPS-2143. The calcilytic prevented the effect of LMWHA on 18pS conductance but did not protect the 4pS conductance. Data are means ± SE, n= 13-15, p<0.001. Significance was determined by 1-way ANOVA and post hoc Tukey test for multiple comparisons.

Journal: bioRxiv

Article Title: Low Molecular Weight Hyaluronan Inhibits Lung Epithelial Ion Channels by Activating the Calcium-Sensing Receptor

doi: 10.1101/2022.09.07.506946

Figure Lengend Snippet: ENaC activity in human AT2 cells in-situ in precision cut lung slices from human lung tissue of a patient (more details are needed). A . Control recording from an ATII cells bathed in Ringer’s solution. Two conductance are visible, a 4pS and an 18 pS, the arrows indicate the open (o) and closed (c) states of the channels. B . Exposure of human lung slices to 150μg/ml LMW-HA for 4hrs reduced channels activities of conductances. C . incubation 1μM NPS-2143 abrogated LMW-HA effect on 18pS conductance but not on 4pS. D and E depict the summary data of both channels activities in control, 4hrs post exposure to 150μg/ml LMW-HA, and 4hrs post incubation with 150μg/ml LMW-HA and 1μM NPS-2143. The calcilytic prevented the effect of LMWHA on 18pS conductance but did not protect the 4pS conductance. Data are means ± SE, n= 13-15, p<0.001. Significance was determined by 1-way ANOVA and post hoc Tukey test for multiple comparisons.

Techniques: Activity Assay, In Situ, Control, Incubation

Figure 1. Phylogenetic tree of P2C-ATPase homologous sequences. Maximum likelihood phylo- genetic tree of P2C-ATPase homologous protein sequences selected from representative species of metazoan, nematodes, fungi, bacteria and plants. Each tree leaf reports the corresponding organism and RefSeq protein accession number. Nodes supported by bootstrap values are indicated by labels.

Journal: International journal of molecular sciences

Article Title: The Non-Gastric H + /K + ATPase (ATP12A) Is Expressed in Mammalian Spermatozoa.

doi: 10.3390/ijms23031048

Figure Lengend Snippet: Figure 1. Phylogenetic tree of P2C-ATPase homologous sequences. Maximum likelihood phylo- genetic tree of P2C-ATPase homologous protein sequences selected from representative species of metazoan, nematodes, fungi, bacteria and plants. Each tree leaf reports the corresponding organism and RefSeq protein accession number. Nodes supported by bootstrap values are indicated by labels.

Article Snippet: Afterward, spermatozoa were incubated for 1 h with different primary antibodies: the polyclonal anti-ATP12A antibody (1:100 dilution) previously adopted in the Western blot analysis; the mouse monoclonal anti-ATP1A1 antibody (1:100 dilution), which recognizes an epitope between amino acid residues 496-506 of the Na+/K+ ATPase α1 subunit (MA3929, Thermo Fisher Scientific, Waltham, MA, USA); the anti-ATP1A4 polyclonal antibody (1:100 dilution), which recognizes the residues 1-90 of the N-terminal region of the α4 subunit isoform of Na+/K+ ATPase (E-AB-65236, Elabscience, Houston, TX, USA); and the anti-ATP1B1 polyclonal antibody (1:100 dilution), which recognizes an epitope between amino acid residues 64-241 of the Na+/K+ ATPase β1 subunit isoform (CSBPA002326LA01HU, CUSABIO, Houston, TX, USA).

Techniques: Bacteria

Amino acid sequences of FXYD3 and its derivatives. (A) Wild-type (WT) FXYD3. Cys residues mutated to Ser in full-length recombinant protein and the FXYD motif in the extracellular domain are shown in red font and are underlined. A Cys residue (indicated by *) in the cytoplasmic domain near the inner membrane leaflet and bracketed by basic amino acids (here Lys) is obligatory for susceptibility to glutathionylation of members of the FXYD protein family. Such susceptibility is critical for a FXYD protein, including FXYD3, to counter glutathionylation of the β1 Na + /K + -ATPase subunit. (B) Amino acid sequence of FXYD3-pep SKSK. Ser residues (indicated by *) that replaced Cys residues in the corresponding sites in WT FXYD3 are indicated in red font. (C) Amino acid sequence of FXYD3-pep CKCK that has Cys residues (indicated by *) of WT FXYD3 retained. (D) Immunoblots for GSH indicating glutathionylation of FXYD3-pep CKCK but not FXYD3-pep SKSK with exposure to GSSG or GSH/H 2 O 2 . (E) Reversibility of FXYD3-pep CKCK glutathionylation with exposure to DTT. (F) Immunoblot for GSH of cell lysate immunoprecipitated for the β1 Na + /K + -ATPase subunit after exposure to Dox. Pre-exposure to FXYD3-pep SKSK augments Dox-induced β1 Na + /K + -ATPase subunit glutathionylation.

Journal: Frontiers in Oncology

Article Title: Displacement of Native FXYD Protein From Na + /K + -ATPase With Novel FXYD Peptide Derivatives: Effects on Doxorubicin Cytotoxicity

doi: 10.3389/fonc.2022.859216

Figure Lengend Snippet: Amino acid sequences of FXYD3 and its derivatives. (A) Wild-type (WT) FXYD3. Cys residues mutated to Ser in full-length recombinant protein and the FXYD motif in the extracellular domain are shown in red font and are underlined. A Cys residue (indicated by *) in the cytoplasmic domain near the inner membrane leaflet and bracketed by basic amino acids (here Lys) is obligatory for susceptibility to glutathionylation of members of the FXYD protein family. Such susceptibility is critical for a FXYD protein, including FXYD3, to counter glutathionylation of the β1 Na + /K + -ATPase subunit. (B) Amino acid sequence of FXYD3-pep SKSK. Ser residues (indicated by *) that replaced Cys residues in the corresponding sites in WT FXYD3 are indicated in red font. (C) Amino acid sequence of FXYD3-pep CKCK that has Cys residues (indicated by *) of WT FXYD3 retained. (D) Immunoblots for GSH indicating glutathionylation of FXYD3-pep CKCK but not FXYD3-pep SKSK with exposure to GSSG or GSH/H 2 O 2 . (E) Reversibility of FXYD3-pep CKCK glutathionylation with exposure to DTT. (F) Immunoblot for GSH of cell lysate immunoprecipitated for the β1 Na + /K + -ATPase subunit after exposure to Dox. Pre-exposure to FXYD3-pep SKSK augments Dox-induced β1 Na + /K + -ATPase subunit glutathionylation.

Article Snippet: The following primary antibodies were used: Na + -K + ATPase α1 subunit (05-369; Merck Millipore, Burlington, MA, USA), Na + -K + -ATPase β1 subunit (A278; Merck Millipore, USA), FXYD3 (OTI1D1; Sigma-Aldrich, AUS), or GSH (recognizing GS-S-proteins purchased from Virogen, Watertown, MA, USA).

Techniques: Recombinant, Residue, Membrane, Sequencing, Western Blot, Immunoprecipitation

Distribution of FXYD3-pep SKSK and its ability to displace FXYD3 from the α1 Na + /K + -ATPase subunit. (A) FXYD3 and Na + /K + -ATPase α1 subunit expression in MCF-7 and MDA-MB-468 breast cancer cells and in BxPC-3 and Panc-1 pancreatic cancer cells. Expression is normalized to expression in human non-cancer MCF-10A cells; GAPDH was the internal loading control. (B) Immunofluorescence showing distribution of TRITC-labeled FXYD3-pep SKSK in BxPC-3 cells. Cells were exposed to 1 µM TRITC-tagged FXYD3-pep SKSK (TRITC-SKSK) (red) for 2 h and fluorescence microscopy performed 24 h after the peptide was washed off. DAPI was used to counterstain the nucleus (blue). One of 5 similar experiments is shown. Fluorescence is predominantly peri-nuclear. Scales are shown in the middle panel. (C) Immunoblot (IB) of α1 Na + /K + -ATPase subunit with WT FXYD3 immunoprecipitant in lysate of BXPC-3 with and without exposure of the cells to 1 μM FXYD3-pep SKSK for 2 h before lysis. (D) Immunoblot of WT FXYD3 with the α1 subunit immunoprecipitant in the lysate from the cells. C, control; TL, total lysate; non-immune IgG (IgG), negative control for IP. The efficiency of the Co-Ip can be estimated by the comparison of β1 subunit expression in the initial total lysate and the unbound supernatant after IP in BxPC-3 cells (data not shown). Approximate binding efficiency was ~90%.

Journal: Frontiers in Oncology

Article Title: Displacement of Native FXYD Protein From Na + /K + -ATPase With Novel FXYD Peptide Derivatives: Effects on Doxorubicin Cytotoxicity

doi: 10.3389/fonc.2022.859216

Figure Lengend Snippet: Distribution of FXYD3-pep SKSK and its ability to displace FXYD3 from the α1 Na + /K + -ATPase subunit. (A) FXYD3 and Na + /K + -ATPase α1 subunit expression in MCF-7 and MDA-MB-468 breast cancer cells and in BxPC-3 and Panc-1 pancreatic cancer cells. Expression is normalized to expression in human non-cancer MCF-10A cells; GAPDH was the internal loading control. (B) Immunofluorescence showing distribution of TRITC-labeled FXYD3-pep SKSK in BxPC-3 cells. Cells were exposed to 1 µM TRITC-tagged FXYD3-pep SKSK (TRITC-SKSK) (red) for 2 h and fluorescence microscopy performed 24 h after the peptide was washed off. DAPI was used to counterstain the nucleus (blue). One of 5 similar experiments is shown. Fluorescence is predominantly peri-nuclear. Scales are shown in the middle panel. (C) Immunoblot (IB) of α1 Na + /K + -ATPase subunit with WT FXYD3 immunoprecipitant in lysate of BXPC-3 with and without exposure of the cells to 1 μM FXYD3-pep SKSK for 2 h before lysis. (D) Immunoblot of WT FXYD3 with the α1 subunit immunoprecipitant in the lysate from the cells. C, control; TL, total lysate; non-immune IgG (IgG), negative control for IP. The efficiency of the Co-Ip can be estimated by the comparison of β1 subunit expression in the initial total lysate and the unbound supernatant after IP in BxPC-3 cells (data not shown). Approximate binding efficiency was ~90%.

Techniques: Expressing, Control, Immunofluorescence, Labeling, Fluorescence, Microscopy, Western Blot, Lysis, Negative Control, Co-Immunoprecipitation Assay, Comparison, Binding Assay

Plasmalemmal Na + /K + -ATPase as a potential target for FXYD3-pep SKSK. (A) MCF-7 cell survival after a 48-h exposure to ouabain in concentrations indicated, with or without co-exposure to 1 μM doxorubicin (Dox). * indicates difference in additive effect of ouabain and Dox at 25 or 50 nM ouabain vs. 70–150 nM ouabain. N = 5 for each ouabain concentration. (B) Electrogenic Na + /K + -ATPase pump currents (I p ) of MCF-7 cells that do, and MDA468 cells that do not, overexpress Na + /K + -ATPase. The trace of membrane current (Im) was recorded in an MCF-7 cell before and after Na + -K + pump activity was eliminated with exposure to K + -free extracellular solution. The inward shift to a near-zero holding current in K + -free solution identifies I p . Currents were sampled with an electronic cursor after electrical noise caused by extracellular solution change had subsided.

Journal: Frontiers in Oncology

Article Title: Displacement of Native FXYD Protein From Na + /K + -ATPase With Novel FXYD Peptide Derivatives: Effects on Doxorubicin Cytotoxicity

doi: 10.3389/fonc.2022.859216

Figure Lengend Snippet: Plasmalemmal Na + /K + -ATPase as a potential target for FXYD3-pep SKSK. (A) MCF-7 cell survival after a 48-h exposure to ouabain in concentrations indicated, with or without co-exposure to 1 μM doxorubicin (Dox). * indicates difference in additive effect of ouabain and Dox at 25 or 50 nM ouabain vs. 70–150 nM ouabain. N = 5 for each ouabain concentration. (B) Electrogenic Na + /K + -ATPase pump currents (I p ) of MCF-7 cells that do, and MDA468 cells that do not, overexpress Na + /K + -ATPase. The trace of membrane current (Im) was recorded in an MCF-7 cell before and after Na + -K + pump activity was eliminated with exposure to K + -free extracellular solution. The inward shift to a near-zero holding current in K + -free solution identifies I p . Currents were sampled with an electronic cursor after electrical noise caused by extracellular solution change had subsided.

Techniques: Concentration Assay, Membrane, Activity Assay

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