β glycerol phosphate  (Millipore)


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  • 99
    Name:
    Glycerol
    Description:

    Catalog Number:
    g5150
    Price:
    None
    Applications:
    Used as carbon source for cultures grown in Terrific Broth and for long-term storage of cultures.
    Buy from Supplier


    Structured Review

    Millipore β glycerol phosphate
    Glycerol

    https://www.bioz.com/result/β glycerol phosphate/product/Millipore
    Average 99 stars, based on 61 article reviews
    Price from $9.99 to $1999.99
    β glycerol phosphate - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Association of Anabolic Effect of Calcitriol with Osteoclast-Derived Wnt 10b Secretion"

    Article Title: Association of Anabolic Effect of Calcitriol with Osteoclast-Derived Wnt 10b Secretion

    Journal: Nutrients

    doi: 10.3390/nu10091164

    Wnt10b and Wnt 16 protein expression in calcitriol-treated osteoblasts. After preosteoblast 7F2 cells were stimulated with 100 μg/mL ascorbic acid and 10 mM β-glycerol phosphate, Western blot analysis showed both Wnt 10b and Wnt 16 expression in osteoblast at different doses of calcitriol was not significantly different at the protein level. Actin protein served as a loading control. Data are means ± SD ( n = 3).
    Figure Legend Snippet: Wnt10b and Wnt 16 protein expression in calcitriol-treated osteoblasts. After preosteoblast 7F2 cells were stimulated with 100 μg/mL ascorbic acid and 10 mM β-glycerol phosphate, Western blot analysis showed both Wnt 10b and Wnt 16 expression in osteoblast at different doses of calcitriol was not significantly different at the protein level. Actin protein served as a loading control. Data are means ± SD ( n = 3).

    Techniques Used: Expressing, Western Blot

    2) Product Images from "Association of Anabolic Effect of Calcitriol with Osteoclast-Derived Wnt 10b Secretion"

    Article Title: Association of Anabolic Effect of Calcitriol with Osteoclast-Derived Wnt 10b Secretion

    Journal: Nutrients

    doi: 10.3390/nu10091164

    Wnt10b and Wnt 16 protein expression in calcitriol-treated osteoblasts. After preosteoblast 7F2 cells were stimulated with 100 μg/mL ascorbic acid and 10 mM β-glycerol phosphate, Western blot analysis showed both Wnt 10b and Wnt 16 expression in osteoblast at different doses of calcitriol was not significantly different at the protein level. Actin protein served as a loading control. Data are means ± SD ( n = 3).
    Figure Legend Snippet: Wnt10b and Wnt 16 protein expression in calcitriol-treated osteoblasts. After preosteoblast 7F2 cells were stimulated with 100 μg/mL ascorbic acid and 10 mM β-glycerol phosphate, Western blot analysis showed both Wnt 10b and Wnt 16 expression in osteoblast at different doses of calcitriol was not significantly different at the protein level. Actin protein served as a loading control. Data are means ± SD ( n = 3).

    Techniques Used: Expressing, Western Blot

    3) Product Images from "Dual roles of QOA-8a in antiosteoporosis: a combination of bone anabolic and anti-resorptive effects"

    Article Title: Dual roles of QOA-8a in antiosteoporosis: a combination of bone anabolic and anti-resorptive effects

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2017.63

    Osteogenic effects and signaling involved in stimulating osteoblast differentiation by QOA-8a. (A) The Alizarin Red staining on primary BMMSCs incubated with vehicle (DMSO) or with QOA-8a (2 μmol/L) in the presence of β-glycerol phosphate (10 mmol/L), ascorbate-2-phosphate (50 μg/mL) and dexamethasone (10 −7 mol/L) for 7 d. (B) Effects of QOA-8a on the ALP activity in osteoblast differentiated from primary BMMSCs for 7 d. ALP in aliquots of supernatants from vehicle (DMSO), QOA-8a (0.2, 2 μmol/L) or AS (2 μmol/L) was measured by an ALP activity kit. Mean±SD. n =3. * P
    Figure Legend Snippet: Osteogenic effects and signaling involved in stimulating osteoblast differentiation by QOA-8a. (A) The Alizarin Red staining on primary BMMSCs incubated with vehicle (DMSO) or with QOA-8a (2 μmol/L) in the presence of β-glycerol phosphate (10 mmol/L), ascorbate-2-phosphate (50 μg/mL) and dexamethasone (10 −7 mol/L) for 7 d. (B) Effects of QOA-8a on the ALP activity in osteoblast differentiated from primary BMMSCs for 7 d. ALP in aliquots of supernatants from vehicle (DMSO), QOA-8a (0.2, 2 μmol/L) or AS (2 μmol/L) was measured by an ALP activity kit. Mean±SD. n =3. * P

    Techniques Used: Staining, Incubation, ALP Assay, Activity Assay

    4) Product Images from "Phosphate is a specific signal for induction of osteopontin gene expression"

    Article Title: Phosphate is a specific signal for induction of osteopontin gene expression

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Schematic representation of the coordination of alkaline phosphatase activity and osteopontin levels in osteoblasts. Alkaline phosphatase (ALP) activity is induced by ascorbic acid (AA) in a process that requires the p300/CBP and pRB families. ALP then is localized to the plasma membrane and oriented such that its catalytic subunit is ectoplasmic. Within the extracellular environment, ALP cleaves a phosphate (P) from β-glycerol phosphate (βGP) to leave free glycerol (G). The free phosphate enters the cell through a Na-dependent phosphate transporter, which is subject to inhibition by foscarnet. Transport of the phosphate results in the up-regulation of osteopontin (OPN) RNA levels. During the differentiation process, the requirement for the pRB and p300 families ensures that induction of the early differentiation marker, alkaline phosphatase, does not begin until proliferation has shut down, and expression of osteopontin is not induced until alkaline phosphatase is active at the cell surface.
    Figure Legend Snippet: Schematic representation of the coordination of alkaline phosphatase activity and osteopontin levels in osteoblasts. Alkaline phosphatase (ALP) activity is induced by ascorbic acid (AA) in a process that requires the p300/CBP and pRB families. ALP then is localized to the plasma membrane and oriented such that its catalytic subunit is ectoplasmic. Within the extracellular environment, ALP cleaves a phosphate (P) from β-glycerol phosphate (βGP) to leave free glycerol (G). The free phosphate enters the cell through a Na-dependent phosphate transporter, which is subject to inhibition by foscarnet. Transport of the phosphate results in the up-regulation of osteopontin (OPN) RNA levels. During the differentiation process, the requirement for the pRB and p300 families ensures that induction of the early differentiation marker, alkaline phosphatase, does not begin until proliferation has shut down, and expression of osteopontin is not induced until alkaline phosphatase is active at the cell surface.

    Techniques Used: Activity Assay, ALP Assay, Inhibition, Marker, Expressing

    Northern blot analysis of osteopontin expression after addition of exogenous alkaline phosphatase (ExALP) or sodium phosphate. ( A ) The 12S.WT.G1 cell line was cultured for 21 days with growth medium supplemented with ExALP, β-glycerol phosphate (βGP), or ascorbic acid (AA) as stated above each lane. ( B ) The 12S.WT.G1 and parental (MC3T3-E1) cell lines were cultured with growth medium and AA, supplemented with ExALP and βGP or a solution in which ExALP and βGP were incubated and then boiled to inactive the enzyme (Boiled), as stated above each lane. ( C ) The 12S.WT.G1 cell line was cultured for 21 days in the presence of AA and in the presence or absence of 10 mM sodium phosphate (NaPO 4 ). Blots were hybridized sequentially with cDNA probes to osteopontin (OPN) and β-actin.
    Figure Legend Snippet: Northern blot analysis of osteopontin expression after addition of exogenous alkaline phosphatase (ExALP) or sodium phosphate. ( A ) The 12S.WT.G1 cell line was cultured for 21 days with growth medium supplemented with ExALP, β-glycerol phosphate (βGP), or ascorbic acid (AA) as stated above each lane. ( B ) The 12S.WT.G1 and parental (MC3T3-E1) cell lines were cultured with growth medium and AA, supplemented with ExALP and βGP or a solution in which ExALP and βGP were incubated and then boiled to inactive the enzyme (Boiled), as stated above each lane. ( C ) The 12S.WT.G1 cell line was cultured for 21 days in the presence of AA and in the presence or absence of 10 mM sodium phosphate (NaPO 4 ). Blots were hybridized sequentially with cDNA probes to osteopontin (OPN) and β-actin.

    Techniques Used: Northern Blot, Expressing, Cell Culture, Incubation

    Related Articles

    Incubation:

    Article Title: Efficient Single-Strand Break Repair Requires Binding to Both Poly(ADP-Ribose) and DNA by the Central BRCT Domain of XRCC1
    Article Snippet: .. The adsorbed proteins were mock ribosylated in the absence of NAD+ or ribosylated in the presence of the 50 mM NAD+ (Sigma) in PARP1 reaction buffer (50 mM Tris–HCl pH7.5, 0.8 mM MgCl2 , 1% glycerol and 1.5 mM DTT) containing 40 nM single-stranded oligodeoxyribonucleotide (5′-CATATGCCGGAGATCCGCCTCC-3′) and 5 nM human PARP1 (Trevigen) in a final volume of 50 μL at room temp for 30 min. After rinsing (4 × ) with 50 μL of 0.1% Tween 20 in PBS, 50 μL of His-SUMO-XRCC-BRCT1 or its variants (diluted to 25 nM in 20 mM Tris pH7.5, 130 nM NaCl) were added to the adsorbed proteins and incubated on ice for 30 min. .. The wells were then rinsed (4 × ) as above and incubated with 50 μL mouse anti-polyhistidine (His-tag) Mab (Takara Bio, diluted 1:2500 in 20 mM Tris pH7.5, 130 nM NaCl) followed by 50 μL HRP-conjugated mouse anti-mouse IgG (ECL, GE Healthcare, 1: 5000 in dilution buffer) for 30 min each on ice.

    other:

    Article Title: Induction of ANGPTL4 expression in human airway smooth muscle cells by PMA through activation of PKC and MAPK pathways
    Article Snippet: 1,2-Dioctanoyl- sn -glycerol (DOG), 4α-phorbol-12,13-didecanoate (PDD), Gö6983, bisindolylmaleimide I (BIM I or GF109203X), Gö6976, bryostatin 1 and 2, PD169316, SB203580, and manumycin A were purchased from Calbiochem (La Jolla, CA).

    Article Title: Accelerated blood clearance phenomenon upon cross-administration of PEGylated nanocarriers in beagle dogs
    Article Snippet: Glycerin monostearate (GMS) and glycerol distearate (GDS) were supplied by Sigma-Aldrich (St Louis, MO, USA).

    Cell Culture:

    Article Title: Antiobesity effects of the water-soluble fraction of the ethanol extract of Smilax china L. leaf in 3T3-L1 adipocytes
    Article Snippet: .. Measuring glycerol release Differentiated 3T3-L1 adipocytes were cultured with 0.1 or 0.25 mg/mL of wsSCLE and 10 µM of the PKA inhibitor H89 (Sigma-Aldrich, St. Louis, USA) for 24 h. This medium was used in measurement of glycerol release level. ..

    Purification:

    Article Title: Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics
    Article Snippet: .. Saporin-KQ (details for purification in ) was chemically conjugated to the reporter RTA (buffered aqueous glycerol solution, Sigma-Aldrich, Steinheim, Germany) by the same chemical procedure described before in the case of the saporin-HRP conjugate. ..

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  • 99
    Millipore cell permeable gsk 3β peptide inhibitor l803 mts
    <t>GSK-3β-mediated</t> tumor suppression and chemosensitization can be overridden by expression of mutated Mcl-1. (A) GSK-3β-WT, GSK-3β-CA, or GSK-3β-KD was transfected by electroporation into MCF-7 or Her-2-expressing MCF-7 cells, and the percentages of apoptotic cells were then determined by FACS as described in Materials and Methods. (B) GSK-3β-CA was cotransfected with Mcl-1-WT or Mcl-1-CA into MCF-7 cells, and the percentages of apoptotic cells were then determined by FACS. (C) GSK-3β-CA was cotransfected with Mcl-1-WT or Mcl-1-CA into MCF-7 cells, and the percentages of apoptotic cells were then detected by TUNEL assay. (D) GSK-3β-CA was cotransfected with Mcl-1-WT or Mcl-1-CA into MCF-7 cells, and the cell lysates were then analyzed for caspase 9 and PARP. (E) Tumor-bearing mice were treated with intratumor injection of the liposome complex with GSK-3β, and the tumor volumes were then measured twice a week (* and # indicate that the differences between the group of Mcl-1-WT and the vector control with the group of Mcl-1-3A mutant are statistically significant [ P
    Cell Permeable Gsk 3β Peptide Inhibitor L803 Mts, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cell permeable gsk 3β peptide inhibitor l803 mts/product/Millipore
    Average 99 stars, based on 1 article reviews
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    85
    Millipore pkcα β pseudosubstrate
    Phosphorylation of caspase 9 (Casp-9) at Ser144 by PKCζ. (A) Recombinant wild-type (wt) or S144A mutant His 6 -caspase 9 (C287A) was incubated with recombinant His-PKCζ. (B) HEK293 cells were transfected for 24 h with inactive (C287A) wild-type or S144A caspase 9 and constitutively active PKCζ (PKCζ A119E ) or empty vector (v). (C) Recombinant His-caspase 9 was incubated in HeLa cytosolic extract treated with 1 μM OA in the presence of PKCζ pseudosubstrate inhibitor (PSζ) or <t>PKCα/β</t> pseudosubstrate inhibitor (PSα/β) at the concentrations shown. (D) Effects of kinase inhibitors on Ser144 and Thr125 phosphorylation in HeLa cytosolic extract treated with 1 μM OA, 5 μM Ro318220, 10 μM Bis-1, 10 μM Go6976, 10 μM Go6983, or 10 μM pseudosubstrate inhibitor (PS). In each case, samples were analyzed by SDS-PAGE and Western blotting with the indicated antibodies.
    Pkcα β Pseudosubstrate, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pkcα β pseudosubstrate/product/Millipore
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    99
    Millipore β glycerol phosphate
    Phosphorylation of caspase 9 (Casp-9) at Ser144 by PKCζ. (A) Recombinant wild-type (wt) or S144A mutant His 6 -caspase 9 (C287A) was incubated with recombinant His-PKCζ. (B) HEK293 cells were transfected for 24 h with inactive (C287A) wild-type or S144A caspase 9 and constitutively active PKCζ (PKCζ A119E ) or empty vector (v). (C) Recombinant His-caspase 9 was incubated in HeLa cytosolic extract treated with 1 μM OA in the presence of PKCζ pseudosubstrate inhibitor (PSζ) or <t>PKCα/β</t> pseudosubstrate inhibitor (PSα/β) at the concentrations shown. (D) Effects of kinase inhibitors on Ser144 and Thr125 phosphorylation in HeLa cytosolic extract treated with 1 μM OA, 5 μM Ro318220, 10 μM Bis-1, 10 μM Go6976, 10 μM Go6983, or 10 μM pseudosubstrate inhibitor (PS). In each case, samples were analyzed by SDS-PAGE and Western blotting with the indicated antibodies.
    β Glycerol Phosphate, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β glycerol phosphate/product/Millipore
    Average 99 stars, based on 61 article reviews
    Price from $9.99 to $1999.99
    β glycerol phosphate - by Bioz Stars, 2020-09
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    Image Search Results


    GSK-3β-mediated tumor suppression and chemosensitization can be overridden by expression of mutated Mcl-1. (A) GSK-3β-WT, GSK-3β-CA, or GSK-3β-KD was transfected by electroporation into MCF-7 or Her-2-expressing MCF-7 cells, and the percentages of apoptotic cells were then determined by FACS as described in Materials and Methods. (B) GSK-3β-CA was cotransfected with Mcl-1-WT or Mcl-1-CA into MCF-7 cells, and the percentages of apoptotic cells were then determined by FACS. (C) GSK-3β-CA was cotransfected with Mcl-1-WT or Mcl-1-CA into MCF-7 cells, and the percentages of apoptotic cells were then detected by TUNEL assay. (D) GSK-3β-CA was cotransfected with Mcl-1-WT or Mcl-1-CA into MCF-7 cells, and the cell lysates were then analyzed for caspase 9 and PARP. (E) Tumor-bearing mice were treated with intratumor injection of the liposome complex with GSK-3β, and the tumor volumes were then measured twice a week (* and # indicate that the differences between the group of Mcl-1-WT and the vector control with the group of Mcl-1-3A mutant are statistically significant [ P

    Journal: Molecular and Cellular Biology

    Article Title: Degradation of Mcl-1 by ?-TrCP Mediates Glycogen Synthase Kinase 3-Induced Tumor Suppression and Chemosensitization ▿Degradation of Mcl-1 by ?-TrCP Mediates Glycogen Synthase Kinase 3-Induced Tumor Suppression and Chemosensitization ▿ †

    doi: 10.1128/MCB.00620-06

    Figure Lengend Snippet: GSK-3β-mediated tumor suppression and chemosensitization can be overridden by expression of mutated Mcl-1. (A) GSK-3β-WT, GSK-3β-CA, or GSK-3β-KD was transfected by electroporation into MCF-7 or Her-2-expressing MCF-7 cells, and the percentages of apoptotic cells were then determined by FACS as described in Materials and Methods. (B) GSK-3β-CA was cotransfected with Mcl-1-WT or Mcl-1-CA into MCF-7 cells, and the percentages of apoptotic cells were then determined by FACS. (C) GSK-3β-CA was cotransfected with Mcl-1-WT or Mcl-1-CA into MCF-7 cells, and the percentages of apoptotic cells were then detected by TUNEL assay. (D) GSK-3β-CA was cotransfected with Mcl-1-WT or Mcl-1-CA into MCF-7 cells, and the cell lysates were then analyzed for caspase 9 and PARP. (E) Tumor-bearing mice were treated with intratumor injection of the liposome complex with GSK-3β, and the tumor volumes were then measured twice a week (* and # indicate that the differences between the group of Mcl-1-WT and the vector control with the group of Mcl-1-3A mutant are statistically significant [ P

    Article Snippet: The GSK-3β inhibitor TDZD8 and cell-permeable GSK-3β peptide inhibitor L803-mts were purchased from Calbiochem (San Diego, CA).

    Techniques: Expressing, Transfection, Electroporation, FACS, TUNEL Assay, Mouse Assay, Injection, Plasmid Preparation, Mutagenesis

    ). Mcl-1 ubiquitination was examined by anti-His-ubiquitin immunoblotting. (C) Inducible Mule shRNA U 2 OS cells were transfected with siRNA against GSK-3β or β-TrCP for 48 h or treated with 2 μg/ml tetracycline to induce Mule shRNA for 48 h and then stimulated with UV irradiation and staurosporine for 6 h. The phosphorylation status of GSK-3β, β-TrCP, and Mule and the Mcl-1 level were determined in cell lysates by Western blotting. (D) Cells were treated with MG132, LiCl, staurosporine, or UV irradiation as indicated, and the cell lysates were then blotted with specific phospho-Mcl-1 antibodies. (E) Inducible Mule shRNA U 2 OS cells treated with 2 μg/ml tetracycline to induce Mule shRNA for 48 h were transfected with GSK-3β-WT or GSK-3β-KD, and the Mcl-1 level in cell lysates was then determined by Western blotting. (F) In 293T cells, the Myc-Mcl-1 WT or 3A mutant was cotransfected with HA-GSK-3β-WT or Flag-Mule for 48 h, and the Mcl-1 level in cell lysates was then determined by Western blotting.

    Journal: Molecular and Cellular Biology

    Article Title: Degradation of Mcl-1 by ?-TrCP Mediates Glycogen Synthase Kinase 3-Induced Tumor Suppression and Chemosensitization ▿Degradation of Mcl-1 by ?-TrCP Mediates Glycogen Synthase Kinase 3-Induced Tumor Suppression and Chemosensitization ▿ †

    doi: 10.1128/MCB.00620-06

    Figure Lengend Snippet: ). Mcl-1 ubiquitination was examined by anti-His-ubiquitin immunoblotting. (C) Inducible Mule shRNA U 2 OS cells were transfected with siRNA against GSK-3β or β-TrCP for 48 h or treated with 2 μg/ml tetracycline to induce Mule shRNA for 48 h and then stimulated with UV irradiation and staurosporine for 6 h. The phosphorylation status of GSK-3β, β-TrCP, and Mule and the Mcl-1 level were determined in cell lysates by Western blotting. (D) Cells were treated with MG132, LiCl, staurosporine, or UV irradiation as indicated, and the cell lysates were then blotted with specific phospho-Mcl-1 antibodies. (E) Inducible Mule shRNA U 2 OS cells treated with 2 μg/ml tetracycline to induce Mule shRNA for 48 h were transfected with GSK-3β-WT or GSK-3β-KD, and the Mcl-1 level in cell lysates was then determined by Western blotting. (F) In 293T cells, the Myc-Mcl-1 WT or 3A mutant was cotransfected with HA-GSK-3β-WT or Flag-Mule for 48 h, and the Mcl-1 level in cell lysates was then determined by Western blotting.

    Article Snippet: The GSK-3β inhibitor TDZD8 and cell-permeable GSK-3β peptide inhibitor L803-mts were purchased from Calbiochem (San Diego, CA).

    Techniques: shRNA, Transfection, Irradiation, Western Blot, Mutagenesis

    GSK-3β phosphorylates Mcl-1. (A) GSK-3β (HA tagged) was transfected into 293T cells, and Mcl-1 or GSK-3β was then immunoprecipitated (IP) from transfected 293T cell lysates (1,000 μg/lane) and subjected to Western blotting. IgG, immunoglobulin G. (B) GSK-3β associates with Mcl-1 in vivo. Endogenous GSK-3β and Mcl-1 were immunoprecipitated from the breast cancer cell line MDA-MB-453 and subjected to Western blotting. (C) Comparison of the amino acid sequences of the GSK-3β phosphorylation motif in Mcl-1 and other known GSK-3β substrates. Mutant GST-Mcl-1 protein was incubated with WT GST-GSK-3β, and the kinase assay was performed as described in Materials and Methods. (D) WT or 3A mutant GST-Mcl-1 protein was incubated with WT or kinase-dead GST-GSK-3β protein or active Erk2 as indicated, the kinase assay was performed as described in Materials and Methods, and reaction mixture samples were then subjected to SDS-PAGE and blotted with specific phospho-Mcl-1 antibodies. (E) 293T cells were cotransfected with Mcl-1 and GSK-3β-KD or GSK-3β-CA and, 36 h later, were treated with MG132 alone or with LiCl for 10 h. Cell lysates were then blotted with specific phospho-Mcl-1 antibodies. (F) Cells were treated with MG132 alone (10 μM) or with LiCl (20 mM) for 10 h as indicated (cells are still viable under this condition), and cell lysates were then blotted with specific phospho-Mcl-1 antibodies.

    Journal: Molecular and Cellular Biology

    Article Title: Degradation of Mcl-1 by ?-TrCP Mediates Glycogen Synthase Kinase 3-Induced Tumor Suppression and Chemosensitization ▿Degradation of Mcl-1 by ?-TrCP Mediates Glycogen Synthase Kinase 3-Induced Tumor Suppression and Chemosensitization ▿ †

    doi: 10.1128/MCB.00620-06

    Figure Lengend Snippet: GSK-3β phosphorylates Mcl-1. (A) GSK-3β (HA tagged) was transfected into 293T cells, and Mcl-1 or GSK-3β was then immunoprecipitated (IP) from transfected 293T cell lysates (1,000 μg/lane) and subjected to Western blotting. IgG, immunoglobulin G. (B) GSK-3β associates with Mcl-1 in vivo. Endogenous GSK-3β and Mcl-1 were immunoprecipitated from the breast cancer cell line MDA-MB-453 and subjected to Western blotting. (C) Comparison of the amino acid sequences of the GSK-3β phosphorylation motif in Mcl-1 and other known GSK-3β substrates. Mutant GST-Mcl-1 protein was incubated with WT GST-GSK-3β, and the kinase assay was performed as described in Materials and Methods. (D) WT or 3A mutant GST-Mcl-1 protein was incubated with WT or kinase-dead GST-GSK-3β protein or active Erk2 as indicated, the kinase assay was performed as described in Materials and Methods, and reaction mixture samples were then subjected to SDS-PAGE and blotted with specific phospho-Mcl-1 antibodies. (E) 293T cells were cotransfected with Mcl-1 and GSK-3β-KD or GSK-3β-CA and, 36 h later, were treated with MG132 alone or with LiCl for 10 h. Cell lysates were then blotted with specific phospho-Mcl-1 antibodies. (F) Cells were treated with MG132 alone (10 μM) or with LiCl (20 mM) for 10 h as indicated (cells are still viable under this condition), and cell lysates were then blotted with specific phospho-Mcl-1 antibodies.

    Article Snippet: The GSK-3β inhibitor TDZD8 and cell-permeable GSK-3β peptide inhibitor L803-mts were purchased from Calbiochem (San Diego, CA).

    Techniques: Transfection, Immunoprecipitation, Western Blot, In Vivo, Multiple Displacement Amplification, Mutagenesis, Incubation, Kinase Assay, SDS Page

    GSK-3β downregulates Mcl-1. (A) 293T cells transfected with Mcl-1-WT, GSK-3β-WT, GSK-3β-CA, or GSK-3β-KD were treated with the GSK-3β inhibitors LiCl (20 mM), TDZD8 (4 μM), and L803-mts (40 mM), as indicated, for 10 h. Expression of Myc-Mcl-1 and HA-GSK-3β was then analyzed by Western blotting. (B) Lysates of MEF cells and GSK-3β knockout MEF cells were subjected to Western blotting to detect endogenous Mcl-1 and GSK-3β. (C) MEF cells and GSK-3β knockout MEF cells (MEF GSK-3β −/− ) were treated with cycloheximide (20 μM) for the indicated times, and endogenous Mcl-1 was then detected by specific mouse anti-Mcl-1 antibody. Equal amounts of protein were subjected to Western blot analyses, as determined by comparing the amount of tubulin (left). Densitometry results for endogenous Mcl-1 after treatment with cycloheximide were normalized by the intensity of tubulin and then plotted (right), and the half-lives of Mcl-1 were determined. (D) Different kinds of human cancer cell lines were transfected with siRNA against GSK-3β and GSK-3α, and cell lysates were then subjected to Western blotting to detect endogenous Mcl-1. (E) 293T cells transfected with Mcl-1-WT, GSK-3β-WT, GSK-3β-CA, or GSK-3β-KD were treated with or without the proteasome inhibitor MG132 (10 μM) for 10 h and analyzed by Western blotting against Myc-Mcl-1 and HA-GSK-3β.

    Journal: Molecular and Cellular Biology

    Article Title: Degradation of Mcl-1 by ?-TrCP Mediates Glycogen Synthase Kinase 3-Induced Tumor Suppression and Chemosensitization ▿Degradation of Mcl-1 by ?-TrCP Mediates Glycogen Synthase Kinase 3-Induced Tumor Suppression and Chemosensitization ▿ †

    doi: 10.1128/MCB.00620-06

    Figure Lengend Snippet: GSK-3β downregulates Mcl-1. (A) 293T cells transfected with Mcl-1-WT, GSK-3β-WT, GSK-3β-CA, or GSK-3β-KD were treated with the GSK-3β inhibitors LiCl (20 mM), TDZD8 (4 μM), and L803-mts (40 mM), as indicated, for 10 h. Expression of Myc-Mcl-1 and HA-GSK-3β was then analyzed by Western blotting. (B) Lysates of MEF cells and GSK-3β knockout MEF cells were subjected to Western blotting to detect endogenous Mcl-1 and GSK-3β. (C) MEF cells and GSK-3β knockout MEF cells (MEF GSK-3β −/− ) were treated with cycloheximide (20 μM) for the indicated times, and endogenous Mcl-1 was then detected by specific mouse anti-Mcl-1 antibody. Equal amounts of protein were subjected to Western blot analyses, as determined by comparing the amount of tubulin (left). Densitometry results for endogenous Mcl-1 after treatment with cycloheximide were normalized by the intensity of tubulin and then plotted (right), and the half-lives of Mcl-1 were determined. (D) Different kinds of human cancer cell lines were transfected with siRNA against GSK-3β and GSK-3α, and cell lysates were then subjected to Western blotting to detect endogenous Mcl-1. (E) 293T cells transfected with Mcl-1-WT, GSK-3β-WT, GSK-3β-CA, or GSK-3β-KD were treated with or without the proteasome inhibitor MG132 (10 μM) for 10 h and analyzed by Western blotting against Myc-Mcl-1 and HA-GSK-3β.

    Article Snippet: The GSK-3β inhibitor TDZD8 and cell-permeable GSK-3β peptide inhibitor L803-mts were purchased from Calbiochem (San Diego, CA).

    Techniques: Transfection, Expressing, Western Blot, Knock-Out

    Phosphorylation of Mcl-1 is required for Mcl-1 degradation. (A) Mcl-1-WT or Mcl-1-3A was cotransfected with GSK-3β-WT, GSK-3β-CA, or GSK-3β-KD into 293T cells as indicated. Cell lysates were then analyzed by Western blotting against Myc-Mcl-1 and HA-GSK-3β. (B) Mcl-1-WT or Mcl-1-3A was cotransfected with GSK-3β-CA or GSK-3β-KD in MEF or GSK-3β knockout MEF cells (MEF GSK-3β −/− ) as indicated. Cell lysates were analyzed by Western blotting against Myc-Mcl-1 and HA-GSK-3β. (C) Mcl-1-WT or Mcl-1-3A was transfected into 293T cells, and cells were then treated with cycloheximide (CHX) (20 mM) for the indicated times. Cell lysates were analyzed by Western blotting against Myc-Mcl-1. Equal amounts of protein were subjected to Western blot analyses, as determined by comparing amounts of tubulin (left). Densitometry results for Mcl-1-WT and Mcl-1-3A after cycloheximide treatment were plotted, and the half-lives of Mcl-1-WT and Mcl-1-3A were determined (right). (D) 293T cells were treated with cycloheximide (20 μM) or cycloheximide with LiCl (20 mM) for the indicated times. Endogenous Mcl-1 expression was examined by Western blotting, and the half-lives of Mcl-1 were determined as described above (C).

    Journal: Molecular and Cellular Biology

    Article Title: Degradation of Mcl-1 by ?-TrCP Mediates Glycogen Synthase Kinase 3-Induced Tumor Suppression and Chemosensitization ▿Degradation of Mcl-1 by ?-TrCP Mediates Glycogen Synthase Kinase 3-Induced Tumor Suppression and Chemosensitization ▿ †

    doi: 10.1128/MCB.00620-06

    Figure Lengend Snippet: Phosphorylation of Mcl-1 is required for Mcl-1 degradation. (A) Mcl-1-WT or Mcl-1-3A was cotransfected with GSK-3β-WT, GSK-3β-CA, or GSK-3β-KD into 293T cells as indicated. Cell lysates were then analyzed by Western blotting against Myc-Mcl-1 and HA-GSK-3β. (B) Mcl-1-WT or Mcl-1-3A was cotransfected with GSK-3β-CA or GSK-3β-KD in MEF or GSK-3β knockout MEF cells (MEF GSK-3β −/− ) as indicated. Cell lysates were analyzed by Western blotting against Myc-Mcl-1 and HA-GSK-3β. (C) Mcl-1-WT or Mcl-1-3A was transfected into 293T cells, and cells were then treated with cycloheximide (CHX) (20 mM) for the indicated times. Cell lysates were analyzed by Western blotting against Myc-Mcl-1. Equal amounts of protein were subjected to Western blot analyses, as determined by comparing amounts of tubulin (left). Densitometry results for Mcl-1-WT and Mcl-1-3A after cycloheximide treatment were plotted, and the half-lives of Mcl-1-WT and Mcl-1-3A were determined (right). (D) 293T cells were treated with cycloheximide (20 μM) or cycloheximide with LiCl (20 mM) for the indicated times. Endogenous Mcl-1 expression was examined by Western blotting, and the half-lives of Mcl-1 were determined as described above (C).

    Article Snippet: The GSK-3β inhibitor TDZD8 and cell-permeable GSK-3β peptide inhibitor L803-mts were purchased from Calbiochem (San Diego, CA).

    Techniques: Western Blot, Knock-Out, Transfection, Expressing

    β-TrCP mediates Mcl-1 degradation. (A) 293T cells were transfected with the indicated doses of siRNA-β-TrCP or nonspecific siRNA (negative control), and cell lysates were then analyzed for endogenous β-TrCP and Mcl-1. (B) β-TrCP1 or β-TrCP2 (Flag tagged) and Mcl-1 (Myc tagged) were cotransfected into 293T cells, and Mcl-1 and β-TrCP1 or Mcl-1 and β-TrCP2 were then immunoprecipitated (IP) from cell lysates and subjected to Western blotting (IB). (C) Immunocomplexes obtained by β-TrCP1 immunoprecipitation were treated with λ-phosphatase (PPase) and analyzed for Mcl-1. (D) β-TrCP1 (Flag tagged) was cotransfected with Mcl-1-WT or Mcl-1-3A (Myc tagged) into MEF with or without GSK-3β knockout cells, and cells were then treated with MG132 (10 μM) for 10 h. Flag-β-TrCP1 was immunoprecipitated from cell lysates and then analyzed for Mcl-1. (E) Mcl-1 peptide and purified GST-Mcl-1 protein were phosphorylated by GST-GSK-3β and then analyzed with three phospho-Mcl-1 antibodies by dot blotting (left). Immobilized Mcl-1-derived peptides, with or without phosphorylation by GSK-3β, were incubated with [ 35 S]methionine-labeled in vitro-translated (IVT) β-TrCP1 or β-TrCP2 protein and analyzed by autoradiography (right). (F) β-TrCP1 (Flag tagged) was cotransfected with Mcl-1-WT or Mcl-1-3A (Myc tagged) into 293T cells, and cells were then treated with MG132 (10 μM) for 10 h. Flag-β-TrCP1 was immunoprecipitated from cotransfected 293T cell lysates and then analyzed for Mcl-1. IgG, immunoglobulin G. (G) 293T cells were transfected with Mcl-1-WT or Mcl-1-3A and β-TrCP1 or F-box-deleted β-TrCP1 as indicated, and cell lysates were then analyzed for Mcl-1.

    Journal: Molecular and Cellular Biology

    Article Title: Degradation of Mcl-1 by ?-TrCP Mediates Glycogen Synthase Kinase 3-Induced Tumor Suppression and Chemosensitization ▿Degradation of Mcl-1 by ?-TrCP Mediates Glycogen Synthase Kinase 3-Induced Tumor Suppression and Chemosensitization ▿ †

    doi: 10.1128/MCB.00620-06

    Figure Lengend Snippet: β-TrCP mediates Mcl-1 degradation. (A) 293T cells were transfected with the indicated doses of siRNA-β-TrCP or nonspecific siRNA (negative control), and cell lysates were then analyzed for endogenous β-TrCP and Mcl-1. (B) β-TrCP1 or β-TrCP2 (Flag tagged) and Mcl-1 (Myc tagged) were cotransfected into 293T cells, and Mcl-1 and β-TrCP1 or Mcl-1 and β-TrCP2 were then immunoprecipitated (IP) from cell lysates and subjected to Western blotting (IB). (C) Immunocomplexes obtained by β-TrCP1 immunoprecipitation were treated with λ-phosphatase (PPase) and analyzed for Mcl-1. (D) β-TrCP1 (Flag tagged) was cotransfected with Mcl-1-WT or Mcl-1-3A (Myc tagged) into MEF with or without GSK-3β knockout cells, and cells were then treated with MG132 (10 μM) for 10 h. Flag-β-TrCP1 was immunoprecipitated from cell lysates and then analyzed for Mcl-1. (E) Mcl-1 peptide and purified GST-Mcl-1 protein were phosphorylated by GST-GSK-3β and then analyzed with three phospho-Mcl-1 antibodies by dot blotting (left). Immobilized Mcl-1-derived peptides, with or without phosphorylation by GSK-3β, were incubated with [ 35 S]methionine-labeled in vitro-translated (IVT) β-TrCP1 or β-TrCP2 protein and analyzed by autoradiography (right). (F) β-TrCP1 (Flag tagged) was cotransfected with Mcl-1-WT or Mcl-1-3A (Myc tagged) into 293T cells, and cells were then treated with MG132 (10 μM) for 10 h. Flag-β-TrCP1 was immunoprecipitated from cotransfected 293T cell lysates and then analyzed for Mcl-1. IgG, immunoglobulin G. (G) 293T cells were transfected with Mcl-1-WT or Mcl-1-3A and β-TrCP1 or F-box-deleted β-TrCP1 as indicated, and cell lysates were then analyzed for Mcl-1.

    Article Snippet: The GSK-3β inhibitor TDZD8 and cell-permeable GSK-3β peptide inhibitor L803-mts were purchased from Calbiochem (San Diego, CA).

    Techniques: Transfection, Negative Control, Immunoprecipitation, Western Blot, Knock-Out, Purification, Derivative Assay, Incubation, Labeling, In Vitro, Autoradiography

    β-TrCP-associated Mcl-1 turnover mediates GSK-3β-induced apoptosis.

    Journal: Molecular and Cellular Biology

    Article Title: Degradation of Mcl-1 by ?-TrCP Mediates Glycogen Synthase Kinase 3-Induced Tumor Suppression and Chemosensitization ▿Degradation of Mcl-1 by ?-TrCP Mediates Glycogen Synthase Kinase 3-Induced Tumor Suppression and Chemosensitization ▿ †

    doi: 10.1128/MCB.00620-06

    Figure Lengend Snippet: β-TrCP-associated Mcl-1 turnover mediates GSK-3β-induced apoptosis.

    Article Snippet: The GSK-3β inhibitor TDZD8 and cell-permeable GSK-3β peptide inhibitor L803-mts were purchased from Calbiochem (San Diego, CA).

    Techniques:

    Phosphorylation of caspase 9 (Casp-9) at Ser144 by PKCζ. (A) Recombinant wild-type (wt) or S144A mutant His 6 -caspase 9 (C287A) was incubated with recombinant His-PKCζ. (B) HEK293 cells were transfected for 24 h with inactive (C287A) wild-type or S144A caspase 9 and constitutively active PKCζ (PKCζ A119E ) or empty vector (v). (C) Recombinant His-caspase 9 was incubated in HeLa cytosolic extract treated with 1 μM OA in the presence of PKCζ pseudosubstrate inhibitor (PSζ) or PKCα/β pseudosubstrate inhibitor (PSα/β) at the concentrations shown. (D) Effects of kinase inhibitors on Ser144 and Thr125 phosphorylation in HeLa cytosolic extract treated with 1 μM OA, 5 μM Ro318220, 10 μM Bis-1, 10 μM Go6976, 10 μM Go6983, or 10 μM pseudosubstrate inhibitor (PS). In each case, samples were analyzed by SDS-PAGE and Western blotting with the indicated antibodies.

    Journal: Molecular and Cellular Biology

    Article Title: Regulation of Caspase 9 through Phosphorylation by Protein Kinase C Zeta in Response to Hyperosmotic Stress

    doi: 10.1128/MCB.25.23.10543-10555.2005

    Figure Lengend Snippet: Phosphorylation of caspase 9 (Casp-9) at Ser144 by PKCζ. (A) Recombinant wild-type (wt) or S144A mutant His 6 -caspase 9 (C287A) was incubated with recombinant His-PKCζ. (B) HEK293 cells were transfected for 24 h with inactive (C287A) wild-type or S144A caspase 9 and constitutively active PKCζ (PKCζ A119E ) or empty vector (v). (C) Recombinant His-caspase 9 was incubated in HeLa cytosolic extract treated with 1 μM OA in the presence of PKCζ pseudosubstrate inhibitor (PSζ) or PKCα/β pseudosubstrate inhibitor (PSα/β) at the concentrations shown. (D) Effects of kinase inhibitors on Ser144 and Thr125 phosphorylation in HeLa cytosolic extract treated with 1 μM OA, 5 μM Ro318220, 10 μM Bis-1, 10 μM Go6976, 10 μM Go6983, or 10 μM pseudosubstrate inhibitor (PS). In each case, samples were analyzed by SDS-PAGE and Western blotting with the indicated antibodies.

    Article Snippet: Myristoylated PKCζ pseudosubstrate (N-Myr-Ser-Ile-Tyr-Arg-Arg-Gly-Ala-Arg-Arg-Trp-Arg-Lys-Leu), myristoylated PKCα/β pseudosubstrate (N-Myr-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln), and other protein kinase inhibitors were purchased from Calbiochem; acetyl Asp-Glu-Val-Asp 7-amido-4-methylcoumarin (AcDEVD-AMC) was from Biomol; and other reagents were from Sigma.

    Techniques: Recombinant, Mutagenesis, Incubation, Transfection, Plasmid Preparation, SDS Page, Western Blot