l threonine Millipore Search Results


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  • 95
    Millipore l threonine
    Comparison of vitamin B 12 production and growth in E. coli strains expressing Module 4 and 5. a Vitamin B 12 production and growth of E. coli expressing the cobN,S,T genes from a single species ( B. melitensis , S. meliloti or R. capsulatus ) as well as the cobW gene from B. melitensis , S. meliloti , or R. capsulatus . b Vitamin B 12 production and growth of E. coli strains expressing various combinations of the cobN , cobS, cobT , and cobW genes from B. melitensis , S. meliloti , or R. capsulatus . c Vitamin B 12 production and growth of E. coli expressing different variant forms of Module 4. Bm , Sm , Rc , St , and Ec represent abbreviations of strains B. melitensis , S. meliloti , R. capsulatus , S. typhimurium , and E. coli , respectively. 1, 2, 3, 4, and 5 in Fig. 5c represent genes encoding cob(I)yrinic acid a,c-diamide adenosyltransferase, adenosylcobyric acid synthase, L -threonine kinase, <t>threonine-O-3-phosphate</t> decarboxylase, and AdoCbi-P synthase, respectively. All strains were cultured in CM medium for vitamin B 12 production. Error bars indicate standard deviations from triplicate biological replicates
    L Threonine, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 268 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore o phospho l threonine
    Discrimination between different forms of phosphoserine and phosphothreonine by REDOR trajectories REDOR data (circles) for each carbon of <t>O-phospho-L-serine</t> (black), O-phospho-DL-serine (red), O-phospho-L-serine:Ca 2+ (blue) and O-phospho-L-threonine (purple) compared with simulations (solid lines). For the weaker couplings (CO, C α) the REDOR trajectories for each molecule are easily distinguishable from each other and are reproduced well by simulations. Error bars are standard error on the mean of either 6 or 8 replicates. Reduced χ 2 between data and simulation is shown for each trajectory.
    O Phospho L Threonine, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore l a phosphatidylinositol
    Discrimination between different forms of phosphoserine and phosphothreonine by REDOR trajectories REDOR data (circles) for each carbon of <t>O-phospho-L-serine</t> (black), O-phospho-DL-serine (red), O-phospho-L-serine:Ca 2+ (blue) and O-phospho-L-threonine (purple) compared with simulations (solid lines). For the weaker couplings (CO, C α) the REDOR trajectories for each molecule are easily distinguishable from each other and are reproduced well by simulations. Error bars are standard error on the mean of either 6 or 8 replicates. Reduced χ 2 between data and simulation is shown for each trajectory.
    L A Phosphatidylinositol, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Millipore n tert butoxycarbonyl l threonine methyl ester
    Discrimination between different forms of phosphoserine and phosphothreonine by REDOR trajectories REDOR data (circles) for each carbon of <t>O-phospho-L-serine</t> (black), O-phospho-DL-serine (red), O-phospho-L-serine:Ca 2+ (blue) and O-phospho-L-threonine (purple) compared with simulations (solid lines). For the weaker couplings (CO, C α) the REDOR trajectories for each molecule are easily distinguishable from each other and are reproduced well by simulations. Error bars are standard error on the mean of either 6 or 8 replicates. Reduced χ 2 between data and simulation is shown for each trajectory.
    N Tert Butoxycarbonyl L Threonine Methyl Ester, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore dl threonine
    Discrimination between different forms of phosphoserine and phosphothreonine by REDOR trajectories REDOR data (circles) for each carbon of <t>O-phospho-L-serine</t> (black), O-phospho-DL-serine (red), O-phospho-L-serine:Ca 2+ (blue) and O-phospho-L-threonine (purple) compared with simulations (solid lines). For the weaker couplings (CO, C α) the REDOR trajectories for each molecule are easily distinguishable from each other and are reproduced well by simulations. Error bars are standard error on the mean of either 6 or 8 replicates. Reduced χ 2 between data and simulation is shown for each trajectory.
    Dl Threonine, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore phosphorylated threonine
    Tau phosphorylation at T212/S214 in G2019S neurons. A , Western blot analysis of adult brain lysates of the elav-GAL4 control or elav-GAL4 -driving LRRK2 , G2019S , or G2019S-K1096M expressions to detect phospho-tau levels by antibodies AT270 (recognizing pT175/pT181), <t>AT8</t> (pS202/pT205), and AT100 (pT212/pS214). Tau 1 detects total tau protein and α-Tubulin (α-Tub) as control. The immunoreactivities of AT100 are shown as a percentage relative to those of Tau 1, averaged from three independent experiments. B–G , Images of dorsal DA dendrites in the A6 segment of third instar larvae with 109(2)80 driving transgenes TauT175/181A ( B ), G2019S + TauT175/181A ( C ), TauT212A ( D ), G2019S + TauT212A ( E ), TauS214A ( F ), or G2019S + TauS214A ( G ). Dendrites are marked by coexpressed mCD8GFP. Scale bar: (in G ) B–G , 50 μm. H , Quantification of dendritic ends for 109(2)80 ( n = 13), G2019S ( n = 12), TauWT ( n = 15), TauWT + G2019S ( n = 8), TauT175/181A ( n = 10), TauT175/181A + G2019S ( n = 8), TauT212A ( n = 10), TauT212A + G2019S ( n = 10), TauS214A ( n = 10), and TauS214A + G2019S ( n = 10). Averages are the mean ± SEM of dendritic ends per 10,000 μm 2 area, and significance is compared by Mann–Whitney test with * p
    Phosphorylated Threonine, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore l allo threonine
    Afadin is not required for proximal-distal patterning in nephron precursors. Localization of segment-specific proteins in E14.5 kidneys from controls ( Afadin fl/fl ) and mutants ( Afadin fl/fl ; Pax3-cre ) are indicated. NCAM delineates the renal vesicle (RV) and S-shaped body (SB). Distal (D), mid- (M) and proximal (P) regions are marked. ( A-B ′) In RVs, <t>WT1</t> (green) is proximal whereas Lef1 (red) is distal in both control and mutant. ( C-D ′) In SBs, WT1 (green) is proximal, whereas Lef1 (red) is mid-SB and distal SB in control and mutant. ( E-H ′) Epha4 (green) is produced in the distal RV and mid-SB in control and mutant. ( I-J ′) Jag1 (red) localizes to mid-SB in control and mutant. Results are representative of sections from at least two mice. Scale bars: 10 μm.
    L Allo Threonine, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore o phospho dl threonine
    Afadin is not required for proximal-distal patterning in nephron precursors. Localization of segment-specific proteins in E14.5 kidneys from controls ( Afadin fl/fl ) and mutants ( Afadin fl/fl ; Pax3-cre ) are indicated. NCAM delineates the renal vesicle (RV) and S-shaped body (SB). Distal (D), mid- (M) and proximal (P) regions are marked. ( A-B ′) In RVs, <t>WT1</t> (green) is proximal whereas Lef1 (red) is distal in both control and mutant. ( C-D ′) In SBs, WT1 (green) is proximal, whereas Lef1 (red) is mid-SB and distal SB in control and mutant. ( E-H ′) Epha4 (green) is produced in the distal RV and mid-SB in control and mutant. ( I-J ′) Jag1 (red) localizes to mid-SB in control and mutant. Results are representative of sections from at least two mice. Scale bars: 10 μm.
    O Phospho Dl Threonine, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore threonine 41 residues
    Afadin is not required for proximal-distal patterning in nephron precursors. Localization of segment-specific proteins in E14.5 kidneys from controls ( Afadin fl/fl ) and mutants ( Afadin fl/fl ; Pax3-cre ) are indicated. NCAM delineates the renal vesicle (RV) and S-shaped body (SB). Distal (D), mid- (M) and proximal (P) regions are marked. ( A-B ′) In RVs, <t>WT1</t> (green) is proximal whereas Lef1 (red) is distal in both control and mutant. ( C-D ′) In SBs, WT1 (green) is proximal, whereas Lef1 (red) is mid-SB and distal SB in control and mutant. ( E-H ′) Epha4 (green) is produced in the distal RV and mid-SB in control and mutant. ( I-J ′) Jag1 (red) localizes to mid-SB in control and mutant. Results are representative of sections from at least two mice. Scale bars: 10 μm.
    Threonine 41 Residues, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore threonine phosphopeptide
    Afadin is not required for proximal-distal patterning in nephron precursors. Localization of segment-specific proteins in E14.5 kidneys from controls ( Afadin fl/fl ) and mutants ( Afadin fl/fl ; Pax3-cre ) are indicated. NCAM delineates the renal vesicle (RV) and S-shaped body (SB). Distal (D), mid- (M) and proximal (P) regions are marked. ( A-B ′) In RVs, <t>WT1</t> (green) is proximal whereas Lef1 (red) is distal in both control and mutant. ( C-D ′) In SBs, WT1 (green) is proximal, whereas Lef1 (red) is mid-SB and distal SB in control and mutant. ( E-H ′) Epha4 (green) is produced in the distal RV and mid-SB in control and mutant. ( I-J ′) Jag1 (red) localizes to mid-SB in control and mutant. Results are representative of sections from at least two mice. Scale bars: 10 μm.
    Threonine Phosphopeptide, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Comparison of vitamin B 12 production and growth in E. coli strains expressing Module 4 and 5. a Vitamin B 12 production and growth of E. coli expressing the cobN,S,T genes from a single species ( B. melitensis , S. meliloti or R. capsulatus ) as well as the cobW gene from B. melitensis , S. meliloti , or R. capsulatus . b Vitamin B 12 production and growth of E. coli strains expressing various combinations of the cobN , cobS, cobT , and cobW genes from B. melitensis , S. meliloti , or R. capsulatus . c Vitamin B 12 production and growth of E. coli expressing different variant forms of Module 4. Bm , Sm , Rc , St , and Ec represent abbreviations of strains B. melitensis , S. meliloti , R. capsulatus , S. typhimurium , and E. coli , respectively. 1, 2, 3, 4, and 5 in Fig. 5c represent genes encoding cob(I)yrinic acid a,c-diamide adenosyltransferase, adenosylcobyric acid synthase, L -threonine kinase, threonine-O-3-phosphate decarboxylase, and AdoCbi-P synthase, respectively. All strains were cultured in CM medium for vitamin B 12 production. Error bars indicate standard deviations from triplicate biological replicates

    Journal: Nature Communications

    Article Title: Metabolic engineering of Escherichia coli for de novo biosynthesis of vitamin B12

    doi: 10.1038/s41467-018-07412-6

    Figure Lengend Snippet: Comparison of vitamin B 12 production and growth in E. coli strains expressing Module 4 and 5. a Vitamin B 12 production and growth of E. coli expressing the cobN,S,T genes from a single species ( B. melitensis , S. meliloti or R. capsulatus ) as well as the cobW gene from B. melitensis , S. meliloti , or R. capsulatus . b Vitamin B 12 production and growth of E. coli strains expressing various combinations of the cobN , cobS, cobT , and cobW genes from B. melitensis , S. meliloti , or R. capsulatus . c Vitamin B 12 production and growth of E. coli expressing different variant forms of Module 4. Bm , Sm , Rc , St , and Ec represent abbreviations of strains B. melitensis , S. meliloti , R. capsulatus , S. typhimurium , and E. coli , respectively. 1, 2, 3, 4, and 5 in Fig. 5c represent genes encoding cob(I)yrinic acid a,c-diamide adenosyltransferase, adenosylcobyric acid synthase, L -threonine kinase, threonine-O-3-phosphate decarboxylase, and AdoCbi-P synthase, respectively. All strains were cultured in CM medium for vitamin B 12 production. Error bars indicate standard deviations from triplicate biological replicates

    Article Snippet: ALA, (R)-1-Amino-2-propanol, L -threonine O-3-phosphate, and vitamin B12 were from Sigma-Aldrich (USA).

    Techniques: Expressing, Variant Assay, Cell Culture

    Discrimination between different forms of phosphoserine and phosphothreonine by REDOR trajectories REDOR data (circles) for each carbon of O-phospho-L-serine (black), O-phospho-DL-serine (red), O-phospho-L-serine:Ca 2+ (blue) and O-phospho-L-threonine (purple) compared with simulations (solid lines). For the weaker couplings (CO, C α) the REDOR trajectories for each molecule are easily distinguishable from each other and are reproduced well by simulations. Error bars are standard error on the mean of either 6 or 8 replicates. Reduced χ 2 between data and simulation is shown for each trajectory.

    Journal: Journal of magnetic resonance (San Diego, Calif. : 1997)

    Article Title: 31P-Dephased, 13C-Detected REDOR for NMR Crystallography at Natural Isotopic Abundance

    doi: 10.1016/j.jmr.2017.02.020

    Figure Lengend Snippet: Discrimination between different forms of phosphoserine and phosphothreonine by REDOR trajectories REDOR data (circles) for each carbon of O-phospho-L-serine (black), O-phospho-DL-serine (red), O-phospho-L-serine:Ca 2+ (blue) and O-phospho-L-threonine (purple) compared with simulations (solid lines). For the weaker couplings (CO, C α) the REDOR trajectories for each molecule are easily distinguishable from each other and are reproduced well by simulations. Error bars are standard error on the mean of either 6 or 8 replicates. Reduced χ 2 between data and simulation is shown for each trajectory.

    Article Snippet: O-Phospho-L-threonine was purchased from Sigma Aldrich and recrystallized by vapor diffusion.

    Techniques:

    Tau phosphorylation at T212/S214 in G2019S neurons. A , Western blot analysis of adult brain lysates of the elav-GAL4 control or elav-GAL4 -driving LRRK2 , G2019S , or G2019S-K1096M expressions to detect phospho-tau levels by antibodies AT270 (recognizing pT175/pT181), AT8 (pS202/pT205), and AT100 (pT212/pS214). Tau 1 detects total tau protein and α-Tubulin (α-Tub) as control. The immunoreactivities of AT100 are shown as a percentage relative to those of Tau 1, averaged from three independent experiments. B–G , Images of dorsal DA dendrites in the A6 segment of third instar larvae with 109(2)80 driving transgenes TauT175/181A ( B ), G2019S + TauT175/181A ( C ), TauT212A ( D ), G2019S + TauT212A ( E ), TauS214A ( F ), or G2019S + TauS214A ( G ). Dendrites are marked by coexpressed mCD8GFP. Scale bar: (in G ) B–G , 50 μm. H , Quantification of dendritic ends for 109(2)80 ( n = 13), G2019S ( n = 12), TauWT ( n = 15), TauWT + G2019S ( n = 8), TauT175/181A ( n = 10), TauT175/181A + G2019S ( n = 8), TauT212A ( n = 10), TauT212A + G2019S ( n = 10), TauS214A ( n = 10), and TauS214A + G2019S ( n = 10). Averages are the mean ± SEM of dendritic ends per 10,000 μm 2 area, and significance is compared by Mann–Whitney test with * p

    Journal: The Journal of Neuroscience

    Article Title: LRRK2 G2019S Mutation Induces Dendrite Degeneration through Mislocalization and Phosphorylation of Tau by Recruiting Autoactivated GSK3β

    doi: 10.1523/JNEUROSCI.1737-10.2010

    Figure Lengend Snippet: Tau phosphorylation at T212/S214 in G2019S neurons. A , Western blot analysis of adult brain lysates of the elav-GAL4 control or elav-GAL4 -driving LRRK2 , G2019S , or G2019S-K1096M expressions to detect phospho-tau levels by antibodies AT270 (recognizing pT175/pT181), AT8 (pS202/pT205), and AT100 (pT212/pS214). Tau 1 detects total tau protein and α-Tubulin (α-Tub) as control. The immunoreactivities of AT100 are shown as a percentage relative to those of Tau 1, averaged from three independent experiments. B–G , Images of dorsal DA dendrites in the A6 segment of third instar larvae with 109(2)80 driving transgenes TauT175/181A ( B ), G2019S + TauT175/181A ( C ), TauT212A ( D ), G2019S + TauT212A ( E ), TauS214A ( F ), or G2019S + TauS214A ( G ). Dendrites are marked by coexpressed mCD8GFP. Scale bar: (in G ) B–G , 50 μm. H , Quantification of dendritic ends for 109(2)80 ( n = 13), G2019S ( n = 12), TauWT ( n = 15), TauWT + G2019S ( n = 8), TauT175/181A ( n = 10), TauT175/181A + G2019S ( n = 8), TauT212A ( n = 10), TauT212A + G2019S ( n = 10), TauS214A ( n = 10), and TauS214A + G2019S ( n = 10). Averages are the mean ± SEM of dendritic ends per 10,000 μm 2 area, and significance is compared by Mann–Whitney test with * p

    Article Snippet: Primary antibodies used are against Flag (M2; Sigma), α-Tubulin [Developmental Studies Hybridoma Bank (DSHB)], AT8 (recognizes phosphorylated tau at serine 202 and threonine 205) (Innogenetics), Tau1 (Millipore Bioscience Research Reagents), AT270 (recognizes phosphorylated tau at threonine 175 and tyrosine 181) (Pierce-Endogen), AT100 (recognizes phosphorylated tau at threonine 212 and serine 214) (Innogenetics), GSK-3β (Abcam), phospho-Y216 (recognizes phosphorylated GSK-3 at tyrosine 216) (BD Biosciences), Cdk5 (Abcam), green fluorescent protein (GFP; Invitrogen), dsRed (BD Biosciences), Futsch (22C10; DSHB), BP104 (DSHB), anti-tyrosine hydroxylase (TH; Pel-Freez), anti-5-HT (Sigma), and phalloidin (Jackson ImmunoResearch).

    Techniques: Western Blot, MANN-WHITNEY

    In MDCKI cells, hypotonic low chloride treatment significantly increases levels of total and phosphorylated hNCC at the apical plasma membrane and decreases levels of ubiquitylated hNCC. ( A ) Representative immunoblots of NCC and pT58-NCC in total or biotinylated pools isolated from MDCKI cells under control conditions (Ctrl) or following low chloride (LC) stimulation. ( B ) Representative immunoblots of NCC and pT58-NCC levels in samples immunoprecipitated (anti-FLAG-tag antibody) from the biotinylated pool of MDCKI cells under Ctrl or following LC stimulation. ( C ) Representative immunoblots of NCC and ubiquitylated NCC levels in samples immunoprecipitated (anti-FLAG-tag antibody) from the biotinylated pool of MDCKI cells under Ctr) or following low LC stimulation. ( D ) Semi-quantitative assessment of experiments. Biotin NCC levels are relative to total NCC levels. Biotin pT58-NCC levels are relative to total pT58-NCC levels. Ubiquitylated NCC (ubNCC) levels at the plasma membrane are relative to total NCC levels at the plasma membrane. Data were analyzed using an unpaired Student’s t-test and presented as means ± S.E.M. ( n = 3–6). *Indicates significant difference compared to corresponding Ctrl conditions.

    Journal: Scientific Reports

    Article Title: The thiazide sensitive sodium chloride co-transporter NCC is modulated by site-specific ubiquitylation

    doi: 10.1038/s41598-017-12819-0

    Figure Lengend Snippet: In MDCKI cells, hypotonic low chloride treatment significantly increases levels of total and phosphorylated hNCC at the apical plasma membrane and decreases levels of ubiquitylated hNCC. ( A ) Representative immunoblots of NCC and pT58-NCC in total or biotinylated pools isolated from MDCKI cells under control conditions (Ctrl) or following low chloride (LC) stimulation. ( B ) Representative immunoblots of NCC and pT58-NCC levels in samples immunoprecipitated (anti-FLAG-tag antibody) from the biotinylated pool of MDCKI cells under Ctrl or following LC stimulation. ( C ) Representative immunoblots of NCC and ubiquitylated NCC levels in samples immunoprecipitated (anti-FLAG-tag antibody) from the biotinylated pool of MDCKI cells under Ctr) or following low LC stimulation. ( D ) Semi-quantitative assessment of experiments. Biotin NCC levels are relative to total NCC levels. Biotin pT58-NCC levels are relative to total pT58-NCC levels. Ubiquitylated NCC (ubNCC) levels at the plasma membrane are relative to total NCC levels at the plasma membrane. Data were analyzed using an unpaired Student’s t-test and presented as means ± S.E.M. ( n = 3–6). *Indicates significant difference compared to corresponding Ctrl conditions.

    Article Snippet: Antibodies Rabbit polyclonal antibodies against total NCC (from Dr. Mark Knepper, NIH, Bethesda, Maryland, USA) or ref. (from Dr. Johannes Loffing, Institute of Anatomy, University of Zurich, Switzerland), phosphorylated Threonine-58 NCC (pT58) , FLAG-tag (F7425, Sigma), Actin (A2228, Sigma), Proteasome 20S (ab3325), and a mouse monoclonal antibody against ubiquitin (P4DI, Cell Signaling) were used for immunoblotting.

    Techniques: Western Blot, Isolation, Immunoprecipitation, FLAG-tag

    Mutation of K706, K828, and K909 residues in NCC increases membrane abundance of pT58-NCC when expressed in MDCKI cells. ( A ) Representative immunoblots of pT58-NCC and NCC in immunoprecipitated (anti-FLAG-tag) samples isolated from the apical biotinylated pool of various MDCKI cells under control (Ctrl) or low chloride (LC) conditions. ( B ) Semi-quantitative assessment of pT58-NCC levels at the plasma membrane under Ctrl conditions. Data were analyzed using one-way ANOVA followed by Tukey-Kramer multiple comparison test and presented as means ± S.E.M. ( n = 6–9) *indicates significant difference compared to WT-NCC under Ctrl conditions. ( C ) Semi-quantitative assessment of pT58-NCC levels at the plasma membrane under Ctrl or LC conditions. Data were analyzed using a two-way ANOVA followed by Tukey-Kramer multiple comparison test. Data are means ± S.E.M. ( n = 6–9) *indicates significant difference between LC and Ctrl conditions for individual cell line. # Represents significant difference compared to WT-NCC following LC stimulation.

    Journal: Scientific Reports

    Article Title: The thiazide sensitive sodium chloride co-transporter NCC is modulated by site-specific ubiquitylation

    doi: 10.1038/s41598-017-12819-0

    Figure Lengend Snippet: Mutation of K706, K828, and K909 residues in NCC increases membrane abundance of pT58-NCC when expressed in MDCKI cells. ( A ) Representative immunoblots of pT58-NCC and NCC in immunoprecipitated (anti-FLAG-tag) samples isolated from the apical biotinylated pool of various MDCKI cells under control (Ctrl) or low chloride (LC) conditions. ( B ) Semi-quantitative assessment of pT58-NCC levels at the plasma membrane under Ctrl conditions. Data were analyzed using one-way ANOVA followed by Tukey-Kramer multiple comparison test and presented as means ± S.E.M. ( n = 6–9) *indicates significant difference compared to WT-NCC under Ctrl conditions. ( C ) Semi-quantitative assessment of pT58-NCC levels at the plasma membrane under Ctrl or LC conditions. Data were analyzed using a two-way ANOVA followed by Tukey-Kramer multiple comparison test. Data are means ± S.E.M. ( n = 6–9) *indicates significant difference between LC and Ctrl conditions for individual cell line. # Represents significant difference compared to WT-NCC following LC stimulation.

    Article Snippet: Antibodies Rabbit polyclonal antibodies against total NCC (from Dr. Mark Knepper, NIH, Bethesda, Maryland, USA) or ref. (from Dr. Johannes Loffing, Institute of Anatomy, University of Zurich, Switzerland), phosphorylated Threonine-58 NCC (pT58) , FLAG-tag (F7425, Sigma), Actin (A2228, Sigma), Proteasome 20S (ab3325), and a mouse monoclonal antibody against ubiquitin (P4DI, Cell Signaling) were used for immunoblotting.

    Techniques: Mutagenesis, Western Blot, Immunoprecipitation, FLAG-tag, Isolation

    Afadin is not required for proximal-distal patterning in nephron precursors. Localization of segment-specific proteins in E14.5 kidneys from controls ( Afadin fl/fl ) and mutants ( Afadin fl/fl ; Pax3-cre ) are indicated. NCAM delineates the renal vesicle (RV) and S-shaped body (SB). Distal (D), mid- (M) and proximal (P) regions are marked. ( A-B ′) In RVs, WT1 (green) is proximal whereas Lef1 (red) is distal in both control and mutant. ( C-D ′) In SBs, WT1 (green) is proximal, whereas Lef1 (red) is mid-SB and distal SB in control and mutant. ( E-H ′) Epha4 (green) is produced in the distal RV and mid-SB in control and mutant. ( I-J ′) Jag1 (red) localizes to mid-SB in control and mutant. Results are representative of sections from at least two mice. Scale bars: 10 μm.

    Journal: Development (Cambridge, England)

    Article Title: De novo lumen formation and elongation in the developing nephron: a central role for afadin in apical polarity

    doi: 10.1242/dev.087957

    Figure Lengend Snippet: Afadin is not required for proximal-distal patterning in nephron precursors. Localization of segment-specific proteins in E14.5 kidneys from controls ( Afadin fl/fl ) and mutants ( Afadin fl/fl ; Pax3-cre ) are indicated. NCAM delineates the renal vesicle (RV) and S-shaped body (SB). Distal (D), mid- (M) and proximal (P) regions are marked. ( A-B ′) In RVs, WT1 (green) is proximal whereas Lef1 (red) is distal in both control and mutant. ( C-D ′) In SBs, WT1 (green) is proximal, whereas Lef1 (red) is mid-SB and distal SB in control and mutant. ( E-H ′) Epha4 (green) is produced in the distal RV and mid-SB in control and mutant. ( I-J ′) Jag1 (red) localizes to mid-SB in control and mutant. Results are representative of sections from at least two mice. Scale bars: 10 μm.

    Article Snippet: All primary antibodies were used 1:100 unless stated otherwise: afadin (Sigma, A0224), nectin 1 (MBL, D146-3), nectin 2 (MBL, D083-3), nectin 3 (MBL, D084-3 and a gift from Y. Takai, Osaka University, Japan), NCAM (DSHB, 5B8; GeneTex H28-123) , R-cadherin (1:50, DSHB, MRCD5) , Six2 (1:200, a gift from A. McMahon, Harvard University, MA, USA) , Meis1/2 (Santa Cruz Biotechnology, sc-10599), laminin (Sigma, 9393), Jag1 (Sigma, A37872), Lef1 (Cell Signaling), WT1 (Millipore, 05-753), Epha4 (BD Biosciences, 610471), Par6b (SCBT, sc-67392), Par3 (Millipore, 07-330), aPKCζ (SCBT, C-20) and phalloidin 647 (Invitrogen, 42008A).

    Techniques: Mutagenesis, Produced, Mouse Assay