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  • 99
    Thermo Fisher mops sds running buffer
    CHD5 protein expression in NBLS cells following transient transfection with miRNA mimics A. Western blot analysis of transfected cells with indicated microRNAs. Post transfection, cells were washed twice with PBS and isolated cell extracts as described in methods [ 41 ]. Whole cell extracts (100 μg) either transfected with indicated miRNAs or mock transfected were subjected to polyacrylamide gel electrophoresis (4-12% <t>SDS-PAGE),</t> using NuPAGE Bis-Tris gels with <t>MOPS-SDS</t> Running Buffer Allstars siRNA and miRNA-454 were used as negative controls. Proteins were transferred on to nitrocellulose membranes (GE Healthcare Life Sciences) and probed with antibodies using rabbit polyclonal CHD5 , actin (Santa Cruz Biotechnology, CA 1:1000), rabbit polyclonal CHD4 (Bethyl 1:2000), and MYCN monoclonal (1:5000; BD Biosciences). Almost complete reduction of CHD5 protein levels were observed for miR-20b, miR-93, miR-17, and miR-211 as indicated, but no change in CHD4, actin or MYCN levels were seen. B. Densitometric analysis of CHD5 protein expression in NBLS cell line. The number of pixels from each band was measured, and a bar graph was created using the Prism to indicate the difference in CHD5 expression upon miRNA transfection. Data are expressed as the standard error mean (SEM). Statistical analysis was performed using the Prism one way ANOVA method followed by Tukey's post-test. Statistical significance relative to the control Allstar siRNA is indicated: *p
    Mops Sds Running Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1709 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher nupage mops sds running buffer
    LRRC 8A expression in the plasma membrane of A549 cells. Cells were exposed to isotonic conditions (Control), hypotonic conditions and isotonic conditions in the presence of 100 μ mol L −1 H 2 O 2 or 10 μ mol L −1 cisplatin for 18hs. Proteins with components exposed to the extracellular compartment were biotinylated and cells were lysed. Biotinylated LRRC 8A and Na + /K + ‐ ATP ase were extracted and separated/quantified by <t>SDS</t> ‐ <t>PAGE</t> /western blotting. As the molecular weight of LRRC 8A and Na + /K + ‐ ATP ase are close (94 kD a/110 kD a) western blots were run in parallel on separate gels. (A) Representative blots. Total indicates total cell homogenates prepared from cells not treated with biotin. (B) Protein ratio LRRC 8A/Na + /K + ‐ ATP ase from 3 sets of experiments. *indicates significantly increase compared to isotonic control cells (Student′s t ‐test).
    Nupage Mops Sds Running Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2703 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher 1x nupage mops sds running buffer
    LRRC 8A expression in the plasma membrane of A549 cells. Cells were exposed to isotonic conditions (Control), hypotonic conditions and isotonic conditions in the presence of 100 μ mol L −1 H 2 O 2 or 10 μ mol L −1 cisplatin for 18hs. Proteins with components exposed to the extracellular compartment were biotinylated and cells were lysed. Biotinylated LRRC 8A and Na + /K + ‐ ATP ase were extracted and separated/quantified by <t>SDS</t> ‐ <t>PAGE</t> /western blotting. As the molecular weight of LRRC 8A and Na + /K + ‐ ATP ase are close (94 kD a/110 kD a) western blots were run in parallel on separate gels. (A) Representative blots. Total indicates total cell homogenates prepared from cells not treated with biotin. (B) Protein ratio LRRC 8A/Na + /K + ‐ ATP ase from 3 sets of experiments. *indicates significantly increase compared to isotonic control cells (Student′s t ‐test).
    1x Nupage Mops Sds Running Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mes sds running buffer
    LRRC 8A expression in the plasma membrane of A549 cells. Cells were exposed to isotonic conditions (Control), hypotonic conditions and isotonic conditions in the presence of 100 μ mol L −1 H 2 O 2 or 10 μ mol L −1 cisplatin for 18hs. Proteins with components exposed to the extracellular compartment were biotinylated and cells were lysed. Biotinylated LRRC 8A and Na + /K + ‐ ATP ase were extracted and separated/quantified by <t>SDS</t> ‐ <t>PAGE</t> /western blotting. As the molecular weight of LRRC 8A and Na + /K + ‐ ATP ase are close (94 kD a/110 kD a) western blots were run in parallel on separate gels. (A) Representative blots. Total indicates total cell homogenates prepared from cells not treated with biotin. (B) Protein ratio LRRC 8A/Na + /K + ‐ ATP ase from 3 sets of experiments. *indicates significantly increase compared to isotonic control cells (Student′s t ‐test).
    Mes Sds Running Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1854 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher nupage mops sds buffer
    LRRC 8A expression in the plasma membrane of A549 cells. Cells were exposed to isotonic conditions (Control), hypotonic conditions and isotonic conditions in the presence of 100 μ mol L −1 H 2 O 2 or 10 μ mol L −1 cisplatin for 18hs. Proteins with components exposed to the extracellular compartment were biotinylated and cells were lysed. Biotinylated LRRC 8A and Na + /K + ‐ ATP ase were extracted and separated/quantified by <t>SDS</t> ‐ <t>PAGE</t> /western blotting. As the molecular weight of LRRC 8A and Na + /K + ‐ ATP ase are close (94 kD a/110 kD a) western blots were run in parallel on separate gels. (A) Representative blots. Total indicates total cell homogenates prepared from cells not treated with biotin. (B) Protein ratio LRRC 8A/Na + /K + ‐ ATP ase from 3 sets of experiments. *indicates significantly increase compared to isotonic control cells (Student′s t ‐test).
    Nupage Mops Sds Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher buffer
    LRRC 8A expression in the plasma membrane of A549 cells. Cells were exposed to isotonic conditions (Control), hypotonic conditions and isotonic conditions in the presence of 100 μ mol L −1 H 2 O 2 or 10 μ mol L −1 cisplatin for 18hs. Proteins with components exposed to the extracellular compartment were biotinylated and cells were lysed. Biotinylated LRRC 8A and Na + /K + ‐ ATP ase were extracted and separated/quantified by <t>SDS</t> ‐ <t>PAGE</t> /western blotting. As the molecular weight of LRRC 8A and Na + /K + ‐ ATP ase are close (94 kD a/110 kD a) western blots were run in parallel on separate gels. (A) Representative blots. Total indicates total cell homogenates prepared from cells not treated with biotin. (B) Protein ratio LRRC 8A/Na + /K + ‐ ATP ase from 3 sets of experiments. *indicates significantly increase compared to isotonic control cells (Student′s t ‐test).
    Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 29794 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mops morpholinepropanesulfonic acid sds running buffer
    LRRC 8A expression in the plasma membrane of A549 cells. Cells were exposed to isotonic conditions (Control), hypotonic conditions and isotonic conditions in the presence of 100 μ mol L −1 H 2 O 2 or 10 μ mol L −1 cisplatin for 18hs. Proteins with components exposed to the extracellular compartment were biotinylated and cells were lysed. Biotinylated LRRC 8A and Na + /K + ‐ ATP ase were extracted and separated/quantified by <t>SDS</t> ‐ <t>PAGE</t> /western blotting. As the molecular weight of LRRC 8A and Na + /K + ‐ ATP ase are close (94 kD a/110 kD a) western blots were run in parallel on separate gels. (A) Representative blots. Total indicates total cell homogenates prepared from cells not treated with biotin. (B) Protein ratio LRRC 8A/Na + /K + ‐ ATP ase from 3 sets of experiments. *indicates significantly increase compared to isotonic control cells (Student′s t ‐test).
    Mops Morpholinepropanesulfonic Acid Sds Running Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CHD5 protein expression in NBLS cells following transient transfection with miRNA mimics A. Western blot analysis of transfected cells with indicated microRNAs. Post transfection, cells were washed twice with PBS and isolated cell extracts as described in methods [ 41 ]. Whole cell extracts (100 μg) either transfected with indicated miRNAs or mock transfected were subjected to polyacrylamide gel electrophoresis (4-12% SDS-PAGE), using NuPAGE Bis-Tris gels with MOPS-SDS Running Buffer Allstars siRNA and miRNA-454 were used as negative controls. Proteins were transferred on to nitrocellulose membranes (GE Healthcare Life Sciences) and probed with antibodies using rabbit polyclonal CHD5 , actin (Santa Cruz Biotechnology, CA 1:1000), rabbit polyclonal CHD4 (Bethyl 1:2000), and MYCN monoclonal (1:5000; BD Biosciences). Almost complete reduction of CHD5 protein levels were observed for miR-20b, miR-93, miR-17, and miR-211 as indicated, but no change in CHD4, actin or MYCN levels were seen. B. Densitometric analysis of CHD5 protein expression in NBLS cell line. The number of pixels from each band was measured, and a bar graph was created using the Prism to indicate the difference in CHD5 expression upon miRNA transfection. Data are expressed as the standard error mean (SEM). Statistical analysis was performed using the Prism one way ANOVA method followed by Tukey's post-test. Statistical significance relative to the control Allstar siRNA is indicated: *p

    Journal: Oncotarget

    Article Title: Role of microRNAs in epigenetic silencing of the CHD5 tumor suppressor gene in neuroblastomas

    doi: 10.18632/oncotarget.7434

    Figure Lengend Snippet: CHD5 protein expression in NBLS cells following transient transfection with miRNA mimics A. Western blot analysis of transfected cells with indicated microRNAs. Post transfection, cells were washed twice with PBS and isolated cell extracts as described in methods [ 41 ]. Whole cell extracts (100 μg) either transfected with indicated miRNAs or mock transfected were subjected to polyacrylamide gel electrophoresis (4-12% SDS-PAGE), using NuPAGE Bis-Tris gels with MOPS-SDS Running Buffer Allstars siRNA and miRNA-454 were used as negative controls. Proteins were transferred on to nitrocellulose membranes (GE Healthcare Life Sciences) and probed with antibodies using rabbit polyclonal CHD5 , actin (Santa Cruz Biotechnology, CA 1:1000), rabbit polyclonal CHD4 (Bethyl 1:2000), and MYCN monoclonal (1:5000; BD Biosciences). Almost complete reduction of CHD5 protein levels were observed for miR-20b, miR-93, miR-17, and miR-211 as indicated, but no change in CHD4, actin or MYCN levels were seen. B. Densitometric analysis of CHD5 protein expression in NBLS cell line. The number of pixels from each band was measured, and a bar graph was created using the Prism to indicate the difference in CHD5 expression upon miRNA transfection. Data are expressed as the standard error mean (SEM). Statistical analysis was performed using the Prism one way ANOVA method followed by Tukey's post-test. Statistical significance relative to the control Allstar siRNA is indicated: *p

    Article Snippet: Western analysis Whole cell extracts (100 μg), either transfected with indicated miRNAs or mock transfected, were subjected to polyacrylamide gel electrophoresis (4-12% SDS-PAGE), using NuPAGE Bis-Tris gels with MOPS-SDS Running Buffer (Invitrogen, Grand Island, NY).

    Techniques: Expressing, Transfection, Western Blot, Isolation, Polyacrylamide Gel Electrophoresis, SDS Page

    Analysis of A244_N332-rgp120 secreted from stable MGAT1 - CHO cell lines. Six stable MGAT1 - CHO cell lines identified with the ClonePix2 were selected as potential substrates for HIV vaccine production. (A) Immunoblot of affinity-purified rgp120 (50 ng per lane) produced by each of six A244_N332-rgp120 cell lines: 3E, 5C, 5D, 3F, 6B, and 5F. Purified A244_N332-rgp120 produced in normal CHO DG44 cells (692) was shown for purpose of comparison. (B) Comparison of A244_N332-rgp120 protein yields as determined by ELISA from the six MGAT1 - CHO cell lines. ( C) SDS PAGE of rgp120 produced by the 5F MGAT1 - CHO cell line. Supernatant samples (10 μ l per lane) collected over the time course of the culture were electrophoresed on a 4–12% NuPage PAGE SDS gel in MOPS buffer (Thermo Scientific, Waltham, MA). The gel was stained with Simply Blue (Thermo Scientific, Waltham, MA) and visualized using an Innotech FluoChem2 system (Genetic Technologies, Grover, MO).

    Journal: PLoS ONE

    Article Title: Robotic selection for the rapid development of stable CHO cell lines for HIV vaccine production

    doi: 10.1371/journal.pone.0197656

    Figure Lengend Snippet: Analysis of A244_N332-rgp120 secreted from stable MGAT1 - CHO cell lines. Six stable MGAT1 - CHO cell lines identified with the ClonePix2 were selected as potential substrates for HIV vaccine production. (A) Immunoblot of affinity-purified rgp120 (50 ng per lane) produced by each of six A244_N332-rgp120 cell lines: 3E, 5C, 5D, 3F, 6B, and 5F. Purified A244_N332-rgp120 produced in normal CHO DG44 cells (692) was shown for purpose of comparison. (B) Comparison of A244_N332-rgp120 protein yields as determined by ELISA from the six MGAT1 - CHO cell lines. ( C) SDS PAGE of rgp120 produced by the 5F MGAT1 - CHO cell line. Supernatant samples (10 μ l per lane) collected over the time course of the culture were electrophoresed on a 4–12% NuPage PAGE SDS gel in MOPS buffer (Thermo Scientific, Waltham, MA). The gel was stained with Simply Blue (Thermo Scientific, Waltham, MA) and visualized using an Innotech FluoChem2 system (Genetic Technologies, Grover, MO).

    Article Snippet: For molecular mass analysis post digestion, 2 μg of each protein was electrophoresed on a 4–12% Bis-Tris PAGE SDS gel in MOPS running buffer, and stained with Coomassie Simply Blue (Thermo Scientific, Waltham, MA).

    Techniques: Affinity Purification, Produced, Purification, Enzyme-linked Immunosorbent Assay, SDS Page, Polyacrylamide Gel Electrophoresis, SDS-Gel, Staining

    ( a ) The trace from a typical size-exclusion run of purified base is shown. The peak between the void and the assembled base contains misassembled base and on a gel looks similar to properly assembled base. ( b ) Gel samples from a typical base purification run on a 10% NuPAGE gel in MOPS buffer and stained using Coomassie blue

    Journal: Methods in molecular biology (Clifton, N.J.)

    Article Title: Recombinant Expression, Unnatural Amino Acid Incorporation, and Site-Specific Labeling of 26S Proteasomal Subcomplexes

    doi: 10.1007/978-1-4939-8706-1_15

    Figure Lengend Snippet: ( a ) The trace from a typical size-exclusion run of purified base is shown. The peak between the void and the assembled base contains misassembled base and on a gel looks similar to properly assembled base. ( b ) Gel samples from a typical base purification run on a 10% NuPAGE gel in MOPS buffer and stained using Coomassie blue

    Article Snippet: 20 × NuPAGE MOPS running buffer (Thermo Fisher).

    Techniques: Purification, Staining

    ( a ) The trace from a typical size-exclusion run of purified lid is shown. The peak after the assembled lid contains mostly cleaved MBP. ( b ) Gel samples from a typical lid purification run on a 10% NuPAGE gel in MOPS buffer and stained using Coomassie blue

    Journal: Methods in molecular biology (Clifton, N.J.)

    Article Title: Recombinant Expression, Unnatural Amino Acid Incorporation, and Site-Specific Labeling of 26S Proteasomal Subcomplexes

    doi: 10.1007/978-1-4939-8706-1_15

    Figure Lengend Snippet: ( a ) The trace from a typical size-exclusion run of purified lid is shown. The peak after the assembled lid contains mostly cleaved MBP. ( b ) Gel samples from a typical lid purification run on a 10% NuPAGE gel in MOPS buffer and stained using Coomassie blue

    Article Snippet: 20 × NuPAGE MOPS running buffer (Thermo Fisher).

    Techniques: Purification, Staining

    LRRC 8A expression in the plasma membrane of A549 cells. Cells were exposed to isotonic conditions (Control), hypotonic conditions and isotonic conditions in the presence of 100 μ mol L −1 H 2 O 2 or 10 μ mol L −1 cisplatin for 18hs. Proteins with components exposed to the extracellular compartment were biotinylated and cells were lysed. Biotinylated LRRC 8A and Na + /K + ‐ ATP ase were extracted and separated/quantified by SDS ‐ PAGE /western blotting. As the molecular weight of LRRC 8A and Na + /K + ‐ ATP ase are close (94 kD a/110 kD a) western blots were run in parallel on separate gels. (A) Representative blots. Total indicates total cell homogenates prepared from cells not treated with biotin. (B) Protein ratio LRRC 8A/Na + /K + ‐ ATP ase from 3 sets of experiments. *indicates significantly increase compared to isotonic control cells (Student′s t ‐test).

    Journal: Physiological Reports

    Article Title: Stress‐induced modulation of volume‐regulated anions channels in human alveolar carcinoma cells . Stress‐induced modulation of volume‐regulated anions channels in human alveolar carcinoma cells

    doi: 10.14814/phy2.13869

    Figure Lengend Snippet: LRRC 8A expression in the plasma membrane of A549 cells. Cells were exposed to isotonic conditions (Control), hypotonic conditions and isotonic conditions in the presence of 100 μ mol L −1 H 2 O 2 or 10 μ mol L −1 cisplatin for 18hs. Proteins with components exposed to the extracellular compartment were biotinylated and cells were lysed. Biotinylated LRRC 8A and Na + /K + ‐ ATP ase were extracted and separated/quantified by SDS ‐ PAGE /western blotting. As the molecular weight of LRRC 8A and Na + /K + ‐ ATP ase are close (94 kD a/110 kD a) western blots were run in parallel on separate gels. (A) Representative blots. Total indicates total cell homogenates prepared from cells not treated with biotin. (B) Protein ratio LRRC 8A/Na + /K + ‐ ATP ase from 3 sets of experiments. *indicates significantly increase compared to isotonic control cells (Student′s t ‐test).

    Article Snippet: Protein samples were mixed with NuPAGE sample buffer containing dithiothreitol (DTT) and proceeded for SDS‐PAGE gel electrophoresis (NuPAGE precast 10% or 4–12% Bis‐tris gels in NuPAGE MOPS SDS running buffer, Invitrogen, Waltham, MA) in NOVEX chambers under reducing/denaturing conditions.

    Techniques: Expressing, SDS Page, Western Blot, Molecular Weight

    Effect of exogenous H 2 O 2 on LRRC 8A mRNA accumulation, LRRC 8A protein expression, swelling induced taurine release, and intracellular signaling associated with apoptosis and cell growth. Taurine release, mRNA accumulation and protein expression in A549 WT were measured by tracer technique, qRT ‐ PCR and SDS ‐ PAGE /western blotting, respectively. (A) Fractional rate constant (min −1 ) for [ 3 H]taurine release was determined in A549 WT cells under isotonic conditions (321 mOsm) and following reduction in the osmolarity to 200 mOsm (shift in tonicity indicated by the arrow) in the absence (control, open circles) or presence of 100 μ mol L −1 H 2 O 2 (acute exposure, closed squares). Alternatively, cells were preexposed to isotonic growth medium containing 100 μ mol L −1 H 2 O 2 for 24 h prior to the release experiment (closed circles). Rate constants at each time point represent mean values from 7 (Control), 4 (preexposed) and 4 (acute exposure) sets of experiments. *indicates that maximal rate constant, obtained 6 min after hypotonic exposure, was significantly different from control cells ( P

    Journal: Physiological Reports

    Article Title: Stress‐induced modulation of volume‐regulated anions channels in human alveolar carcinoma cells . Stress‐induced modulation of volume‐regulated anions channels in human alveolar carcinoma cells

    doi: 10.14814/phy2.13869

    Figure Lengend Snippet: Effect of exogenous H 2 O 2 on LRRC 8A mRNA accumulation, LRRC 8A protein expression, swelling induced taurine release, and intracellular signaling associated with apoptosis and cell growth. Taurine release, mRNA accumulation and protein expression in A549 WT were measured by tracer technique, qRT ‐ PCR and SDS ‐ PAGE /western blotting, respectively. (A) Fractional rate constant (min −1 ) for [ 3 H]taurine release was determined in A549 WT cells under isotonic conditions (321 mOsm) and following reduction in the osmolarity to 200 mOsm (shift in tonicity indicated by the arrow) in the absence (control, open circles) or presence of 100 μ mol L −1 H 2 O 2 (acute exposure, closed squares). Alternatively, cells were preexposed to isotonic growth medium containing 100 μ mol L −1 H 2 O 2 for 24 h prior to the release experiment (closed circles). Rate constants at each time point represent mean values from 7 (Control), 4 (preexposed) and 4 (acute exposure) sets of experiments. *indicates that maximal rate constant, obtained 6 min after hypotonic exposure, was significantly different from control cells ( P

    Article Snippet: Protein samples were mixed with NuPAGE sample buffer containing dithiothreitol (DTT) and proceeded for SDS‐PAGE gel electrophoresis (NuPAGE precast 10% or 4–12% Bis‐tris gels in NuPAGE MOPS SDS running buffer, Invitrogen, Waltham, MA) in NOVEX chambers under reducing/denaturing conditions.

    Techniques: Expressing, Quantitative RT-PCR, SDS Page, Western Blot

    Effect of cisplatin on Akt phosphorylation, swelling‐induced taurine release, and VRAC inactivation. Protein expression was determined by SDS ‐ PAGE /western blotting ( WB ) followed by protein quantification using UN ‐ SCAN ‐ IT . Rate constants and inactivation for the volume‐sensitive taurine release were obtained by tracer technique. (A) Transient Akt activation in A549 cells following exposure to cisplatin and oxaliplatin. Cells were exposed to 20 μ mol L −1 cisplatin or oxaliplatin. Lysates were taken and tested for P‐Akt and total Akt expression at the time points indicated. P‐Akt/Akt expression ratios from 3 sets of experiments were in each setup calculated relative to the untreated controls and presented as mean values ± SEM . Insert: Representative P‐akt and total Akt western blots ( Lane 1 : Control cells; Lanes 2–6: 0.5 , 1, 3, 6, 24 h exposure to cisplatin; Lanes 7–11: 0.5, 1, 3, 6, 24 h exposure to 20 μ mol L −1 oxaliplatin). * and # denote that P‐Akt / Akt expression ratios are statistically different from the ratio in untreated control or the maximal P‐Akt/Akt ratio (time 6 h), respectively (one‐way ANOVA with Fischer's LSD test. (B) A549 cells were exposed for 6 h to Wortmannin (Wort, 5 μ mol L −1 ), HO pic (5 μ mol L −1 ), Rapamycin (Rapa, 400 nmol/L) or BHT (0.5 mmol L −1 ) in the absence or presence of 20 μ mol L −1 cisplatin. Lysates were taken and proceeded for detection of total‐Akt and P‐Akt. Values indicate P‐Akt/Akt expression ratios from 4 sets of experiments were in each setup calculated relative to the untreated controls and presented as mean values ± SEM . * and # denote statistically different from cells not treated with cisplatin and cells treated with cisplatin, respectively ( P

    Journal: Physiological Reports

    Article Title: Stress‐induced modulation of volume‐regulated anions channels in human alveolar carcinoma cells . Stress‐induced modulation of volume‐regulated anions channels in human alveolar carcinoma cells

    doi: 10.14814/phy2.13869

    Figure Lengend Snippet: Effect of cisplatin on Akt phosphorylation, swelling‐induced taurine release, and VRAC inactivation. Protein expression was determined by SDS ‐ PAGE /western blotting ( WB ) followed by protein quantification using UN ‐ SCAN ‐ IT . Rate constants and inactivation for the volume‐sensitive taurine release were obtained by tracer technique. (A) Transient Akt activation in A549 cells following exposure to cisplatin and oxaliplatin. Cells were exposed to 20 μ mol L −1 cisplatin or oxaliplatin. Lysates were taken and tested for P‐Akt and total Akt expression at the time points indicated. P‐Akt/Akt expression ratios from 3 sets of experiments were in each setup calculated relative to the untreated controls and presented as mean values ± SEM . Insert: Representative P‐akt and total Akt western blots ( Lane 1 : Control cells; Lanes 2–6: 0.5 , 1, 3, 6, 24 h exposure to cisplatin; Lanes 7–11: 0.5, 1, 3, 6, 24 h exposure to 20 μ mol L −1 oxaliplatin). * and # denote that P‐Akt / Akt expression ratios are statistically different from the ratio in untreated control or the maximal P‐Akt/Akt ratio (time 6 h), respectively (one‐way ANOVA with Fischer's LSD test. (B) A549 cells were exposed for 6 h to Wortmannin (Wort, 5 μ mol L −1 ), HO pic (5 μ mol L −1 ), Rapamycin (Rapa, 400 nmol/L) or BHT (0.5 mmol L −1 ) in the absence or presence of 20 μ mol L −1 cisplatin. Lysates were taken and proceeded for detection of total‐Akt and P‐Akt. Values indicate P‐Akt/Akt expression ratios from 4 sets of experiments were in each setup calculated relative to the untreated controls and presented as mean values ± SEM . * and # denote statistically different from cells not treated with cisplatin and cells treated with cisplatin, respectively ( P

    Article Snippet: Protein samples were mixed with NuPAGE sample buffer containing dithiothreitol (DTT) and proceeded for SDS‐PAGE gel electrophoresis (NuPAGE precast 10% or 4–12% Bis‐tris gels in NuPAGE MOPS SDS running buffer, Invitrogen, Waltham, MA) in NOVEX chambers under reducing/denaturing conditions.

    Techniques: Expressing, SDS Page, Western Blot, Activation Assay

    Swelling induced release of taurine and LRRC 8A mRNA /protein expression in A549 cells following repetitive hypoosmotic exposure or exposure to varying degrees of hypoosmotic challenge. Taurine release, mRNA accumulation and protein expression, were measured by tracer technique, qRT ‐ PCR and SDS ‐ PAGE /western blotting, respectively. (A) Fractional rate constants (min −1 ) for [ 3 H]taurine release from cisplatin‐sensitive A549 (A549 WT ) cells, preloaded with [ 3 H]taurine, were determined under isotonic/hypotonic conditions and plotted versus time. One group of cells, preincubated in isotonic growth medium, was exposed for 10 min to isotonic Ringer (321 mOsm) before exposure to hypotonic Ringer (321//231 mOsm) (○, n = 10). Another group, adapted for 24 h in hypotonic growth medium (220–226 mOsm), was exposed for 10 min to hypotonic Ringer with a similar tonicity (231 mOsm) followed by an additional hypotonic challenge (231//161 mOsm Ringer) (●, n = 9). Shift in tonicity is indicated by the arrow. Data represent mean values ± SEM . *indicates statistical increased compared to the prehypotonic values ( P

    Journal: Physiological Reports

    Article Title: Stress‐induced modulation of volume‐regulated anions channels in human alveolar carcinoma cells . Stress‐induced modulation of volume‐regulated anions channels in human alveolar carcinoma cells

    doi: 10.14814/phy2.13869

    Figure Lengend Snippet: Swelling induced release of taurine and LRRC 8A mRNA /protein expression in A549 cells following repetitive hypoosmotic exposure or exposure to varying degrees of hypoosmotic challenge. Taurine release, mRNA accumulation and protein expression, were measured by tracer technique, qRT ‐ PCR and SDS ‐ PAGE /western blotting, respectively. (A) Fractional rate constants (min −1 ) for [ 3 H]taurine release from cisplatin‐sensitive A549 (A549 WT ) cells, preloaded with [ 3 H]taurine, were determined under isotonic/hypotonic conditions and plotted versus time. One group of cells, preincubated in isotonic growth medium, was exposed for 10 min to isotonic Ringer (321 mOsm) before exposure to hypotonic Ringer (321//231 mOsm) (○, n = 10). Another group, adapted for 24 h in hypotonic growth medium (220–226 mOsm), was exposed for 10 min to hypotonic Ringer with a similar tonicity (231 mOsm) followed by an additional hypotonic challenge (231//161 mOsm Ringer) (●, n = 9). Shift in tonicity is indicated by the arrow. Data represent mean values ± SEM . *indicates statistical increased compared to the prehypotonic values ( P

    Article Snippet: Protein samples were mixed with NuPAGE sample buffer containing dithiothreitol (DTT) and proceeded for SDS‐PAGE gel electrophoresis (NuPAGE precast 10% or 4–12% Bis‐tris gels in NuPAGE MOPS SDS running buffer, Invitrogen, Waltham, MA) in NOVEX chambers under reducing/denaturing conditions.

    Techniques: Expressing, Quantitative RT-PCR, SDS Page, Western Blot