y-p039  (Echelon Biosciences)


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    Echelon Biosciences y-p039
    Y P039, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    y-p039  (Echelon Biosciences)


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    Echelon Biosciences y-p039
    Y P039, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    polypiposomes  (Echelon Biosciences)


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    Echelon Biosciences polypiposomes
    (A) Binding of tTG to recycling endosomes. The amounts of purified 125 I-tTG bound at 4°C to recycling endosomes immunoisolated from NIH3T3 cells lacking tTG were determined in a gamma counter with all measurements performed in triplicates. Shown are means ± SEM for three independent experiments. (B) The role of phosphoinositides in the tTG interaction with recycling endosomes. Binding of 50 nM purified tTG to recycling endosomes was tested in the presence of 100 µM free phosphoinositides. Bound tTG and endosomal Rab11A/Rab11B and transferrin receptor (TfR) were detected by immuno-blotting. The relative amounts of vesicle-bound tTG were quantified and compared to that in the sample without phosphoinositides in four independent experiments. Bars depict means ± SEM, *p<0.05. (C) The putative phospholipid-binding sequence and site within the fourth domain of tTG. (D) Comparative analysis of tTG binding to phosphoinositides. 125 I-tTG binding to synthetic liposomes <t>(PolyPIPosomes™)</t> containing indicated phiosphoinositides was determined in a gamma counter with all measurements performed in triplicates. Shown are means ± SEM for three independent experiments. (E) Association of tTG with phosphoinositides in cells. tTG was immunoprecipitated from extracts of WI-38 fibroblasts and the immune complexes were probed by immunoblotting for PI(3)P or PI(4)P. (F) The phospholipid-binding site in tTG is required for membrane targeting and externalization of the protein. K598A,K600A,R601A,K602A mutations (m-plbs) were generated within the phospholipid-binding site of tTG. NIH3T3 fibroblasts transfected with wild type tTG (wt) or its mutant deficient in phosphoinositide binding (m-plbs) were left untreated or treated with 10 mM neomycin for 24 h before induction of these proteins for 4 h. The contents of wt and m-plbs tTG in the recycling endosomes and cytosol were defined by immunoblotting ( left panels ). Cell surface and total levels of wt and m-plbs tTG were defined by surface biotinylation, isolation of surface protein fractions, and immunoblotting ( right panels ). The relative contents of wt and m-plbs tTG in the recycling endosomes and on the cell surface were quantified and compared to those for untreated cells expressing wt protein. Shown are representative of three independent experiments. Bars depict means ± SEM, **p<0.005. See also . (G) A proposed mechanism of unconventional tTG secretion. The de novo synthesized intracellular (cytoplasmic) tTG ( hexagons ) does not follow the classical, ER/Golgi-dependent secretion pathway, but is exported via a four-step unconventional secretion process mediated by recycling endosomes ( dotted arrows ). Solid arrows mark the major intracellular recycling route through the PNRC utilized by β1 integrins. See further comments in the text.
    Polypiposomes, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Unconventional Secretion of Tissue Transglutaminase Involves Phospholipid-Dependent Delivery into Recycling Endosomes"

    Article Title: Unconventional Secretion of Tissue Transglutaminase Involves Phospholipid-Dependent Delivery into Recycling Endosomes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0019414

    (A) Binding of tTG to recycling endosomes. The amounts of purified 125 I-tTG bound at 4°C to recycling endosomes immunoisolated from NIH3T3 cells lacking tTG were determined in a gamma counter with all measurements performed in triplicates. Shown are means ± SEM for three independent experiments. (B) The role of phosphoinositides in the tTG interaction with recycling endosomes. Binding of 50 nM purified tTG to recycling endosomes was tested in the presence of 100 µM free phosphoinositides. Bound tTG and endosomal Rab11A/Rab11B and transferrin receptor (TfR) were detected by immuno-blotting. The relative amounts of vesicle-bound tTG were quantified and compared to that in the sample without phosphoinositides in four independent experiments. Bars depict means ± SEM, *p<0.05. (C) The putative phospholipid-binding sequence and site within the fourth domain of tTG. (D) Comparative analysis of tTG binding to phosphoinositides. 125 I-tTG binding to synthetic liposomes (PolyPIPosomes™) containing indicated phiosphoinositides was determined in a gamma counter with all measurements performed in triplicates. Shown are means ± SEM for three independent experiments. (E) Association of tTG with phosphoinositides in cells. tTG was immunoprecipitated from extracts of WI-38 fibroblasts and the immune complexes were probed by immunoblotting for PI(3)P or PI(4)P. (F) The phospholipid-binding site in tTG is required for membrane targeting and externalization of the protein. K598A,K600A,R601A,K602A mutations (m-plbs) were generated within the phospholipid-binding site of tTG. NIH3T3 fibroblasts transfected with wild type tTG (wt) or its mutant deficient in phosphoinositide binding (m-plbs) were left untreated or treated with 10 mM neomycin for 24 h before induction of these proteins for 4 h. The contents of wt and m-plbs tTG in the recycling endosomes and cytosol were defined by immunoblotting ( left panels ). Cell surface and total levels of wt and m-plbs tTG were defined by surface biotinylation, isolation of surface protein fractions, and immunoblotting ( right panels ). The relative contents of wt and m-plbs tTG in the recycling endosomes and on the cell surface were quantified and compared to those for untreated cells expressing wt protein. Shown are representative of three independent experiments. Bars depict means ± SEM, **p<0.005. See also . (G) A proposed mechanism of unconventional tTG secretion. The de novo synthesized intracellular (cytoplasmic) tTG ( hexagons ) does not follow the classical, ER/Golgi-dependent secretion pathway, but is exported via a four-step unconventional secretion process mediated by recycling endosomes ( dotted arrows ). Solid arrows mark the major intracellular recycling route through the PNRC utilized by β1 integrins. See further comments in the text.
    Figure Legend Snippet: (A) Binding of tTG to recycling endosomes. The amounts of purified 125 I-tTG bound at 4°C to recycling endosomes immunoisolated from NIH3T3 cells lacking tTG were determined in a gamma counter with all measurements performed in triplicates. Shown are means ± SEM for three independent experiments. (B) The role of phosphoinositides in the tTG interaction with recycling endosomes. Binding of 50 nM purified tTG to recycling endosomes was tested in the presence of 100 µM free phosphoinositides. Bound tTG and endosomal Rab11A/Rab11B and transferrin receptor (TfR) were detected by immuno-blotting. The relative amounts of vesicle-bound tTG were quantified and compared to that in the sample without phosphoinositides in four independent experiments. Bars depict means ± SEM, *p<0.05. (C) The putative phospholipid-binding sequence and site within the fourth domain of tTG. (D) Comparative analysis of tTG binding to phosphoinositides. 125 I-tTG binding to synthetic liposomes (PolyPIPosomes™) containing indicated phiosphoinositides was determined in a gamma counter with all measurements performed in triplicates. Shown are means ± SEM for three independent experiments. (E) Association of tTG with phosphoinositides in cells. tTG was immunoprecipitated from extracts of WI-38 fibroblasts and the immune complexes were probed by immunoblotting for PI(3)P or PI(4)P. (F) The phospholipid-binding site in tTG is required for membrane targeting and externalization of the protein. K598A,K600A,R601A,K602A mutations (m-plbs) were generated within the phospholipid-binding site of tTG. NIH3T3 fibroblasts transfected with wild type tTG (wt) or its mutant deficient in phosphoinositide binding (m-plbs) were left untreated or treated with 10 mM neomycin for 24 h before induction of these proteins for 4 h. The contents of wt and m-plbs tTG in the recycling endosomes and cytosol were defined by immunoblotting ( left panels ). Cell surface and total levels of wt and m-plbs tTG were defined by surface biotinylation, isolation of surface protein fractions, and immunoblotting ( right panels ). The relative contents of wt and m-plbs tTG in the recycling endosomes and on the cell surface were quantified and compared to those for untreated cells expressing wt protein. Shown are representative of three independent experiments. Bars depict means ± SEM, **p<0.005. See also . (G) A proposed mechanism of unconventional tTG secretion. The de novo synthesized intracellular (cytoplasmic) tTG ( hexagons ) does not follow the classical, ER/Golgi-dependent secretion pathway, but is exported via a four-step unconventional secretion process mediated by recycling endosomes ( dotted arrows ). Solid arrows mark the major intracellular recycling route through the PNRC utilized by β1 integrins. See further comments in the text.

    Techniques Used: Binding Assay, Purification, Sequencing, Immunoprecipitation, Western Blot, Generated, Transfection, Mutagenesis, Isolation, Expressing, Synthesized

    pi3 4 5p 3 polypiposomes  (Echelon Biosciences)


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    Echelon Biosciences pi3 4 5p 3 polypiposomes
    a , Fluorescent IP-WB detects on-site PI4,5P 2 and <t>PI3,4,5P</t> 3 association with epitope-tagged p53 downstream of PIPKIα. FLAG-tagged mutant (R175H) or wild-type p53 were transient-transfected into HEK293FT cells for 24 h. Then the cells were treated with 50 µM PIPKIα inhibitor ISA-2011B (ISA) for 24 h. The ectopically-expressed p53 was IP-ed with FLAG antibody and analyzed by WB using fluorescent antibodies detecting PI4,5P 2 , PI3,4,5P 3 , and p53 simultaneously. N=3. b , Fluorescent IP-WB detects on-site PI4,5P 2 and PI3,4,5P 3 association with endogenous mutant p53 and stress-induced wild-type p53 in a panel of cancer cells. A549 cells expressing wild-type p53 were treated with 30 µM cisplatin or control vehicle for 24 h before being processed for IP against p53. BT-549, GILM2, HS578T, MDA-MB-231, and MDA-MB-468 cells expressing mutant p53 were directly processed for IP against mutant p53. The on-site PI4,5P 2 and PI3,4,5P 3 association with p53 was analyzed simultaneously by WB using fluorescent antibodies. N=3. c , Fluorescent IP-WB detects stress-induced PI3,4,5P 3 association with endogenous mutant p53 downstream of PIPKIα but independent from class I PI3Ks. MDA-MB-231 cells were treated with control vehicle or 30 µM cisplatin with or without the presence of the α-specific PI3K inhibitor alpelisib (10 µM), the pan-PI3K inhibitor buparlisib (2 µM), or the PIPKIα inhibitor ISA-2011B (50 µM) for 24 h. Then the cells were processed for IP against p53 and analyzed by WB using fluorescent antibodies detecting PI3,4,5P 3 and p53 simultaneously. N=3. d-e , PLA of p53-PI4,5P 2 /PI3,4,5P 3 /IPMK/PTEN overlaid with nuclear envelope marker Lamin A/C in MDA-MB-231 cells treated with control vehicle or 30 µM cisplatin for 24 h. The nuclear PLA foci were quantified ( e ). N=30 cells from representative experiments of three repeats. f , IP of IPMK with wild-type and mutant p53 from HCT116 and Cal33 cells respectively treated with 30 µM cisplatin or control vehicle for 24 h. Representative data of three independent experiments are shown. g , IP of PTEN with wild-type and mutant p53 from A549 and MDA-MB-231 cells respectively treated with 30 µM cisplatin or control vehicle for 24 h. Representative data of three independent experiments are shown. h-i , Quantification of the nuclear PLA foci of p53-PI4,5P 2 /PI3,4,5P 3 in MDA-MB-231 cells 48 h after transient transfection with control siRNAs or siRNAs against PIPKIα, IPMK, and PTEN. N=30 cells from representative experiments of three repeats. See PLA images in Extended Data Fig. 5k and knockdown validation by WB in Extended Data Fig. 5l. j , Schematic illustration of p53 in a complex with PI4,5P 2 and PI3,4,5P 3 downstream of PIPKIα and their interconversion by IPMK and PTEN. For all panels, data are represented as mean ±SD, p < 0.01 = **, t test. Scale bar: 5 µm.
    Pi3 4 5p 3 Polypiposomes, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92/100 stars

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    1) Product Images from "A p53-Phosphoinositide Signalosome Regulates Nuclear Akt Activation"

    Article Title: A p53-Phosphoinositide Signalosome Regulates Nuclear Akt Activation

    Journal: bioRxiv

    doi: 10.1101/2021.09.17.460854

    a , Fluorescent IP-WB detects on-site PI4,5P 2 and PI3,4,5P 3 association with epitope-tagged p53 downstream of PIPKIα. FLAG-tagged mutant (R175H) or wild-type p53 were transient-transfected into HEK293FT cells for 24 h. Then the cells were treated with 50 µM PIPKIα inhibitor ISA-2011B (ISA) for 24 h. The ectopically-expressed p53 was IP-ed with FLAG antibody and analyzed by WB using fluorescent antibodies detecting PI4,5P 2 , PI3,4,5P 3 , and p53 simultaneously. N=3. b , Fluorescent IP-WB detects on-site PI4,5P 2 and PI3,4,5P 3 association with endogenous mutant p53 and stress-induced wild-type p53 in a panel of cancer cells. A549 cells expressing wild-type p53 were treated with 30 µM cisplatin or control vehicle for 24 h before being processed for IP against p53. BT-549, GILM2, HS578T, MDA-MB-231, and MDA-MB-468 cells expressing mutant p53 were directly processed for IP against mutant p53. The on-site PI4,5P 2 and PI3,4,5P 3 association with p53 was analyzed simultaneously by WB using fluorescent antibodies. N=3. c , Fluorescent IP-WB detects stress-induced PI3,4,5P 3 association with endogenous mutant p53 downstream of PIPKIα but independent from class I PI3Ks. MDA-MB-231 cells were treated with control vehicle or 30 µM cisplatin with or without the presence of the α-specific PI3K inhibitor alpelisib (10 µM), the pan-PI3K inhibitor buparlisib (2 µM), or the PIPKIα inhibitor ISA-2011B (50 µM) for 24 h. Then the cells were processed for IP against p53 and analyzed by WB using fluorescent antibodies detecting PI3,4,5P 3 and p53 simultaneously. N=3. d-e , PLA of p53-PI4,5P 2 /PI3,4,5P 3 /IPMK/PTEN overlaid with nuclear envelope marker Lamin A/C in MDA-MB-231 cells treated with control vehicle or 30 µM cisplatin for 24 h. The nuclear PLA foci were quantified ( e ). N=30 cells from representative experiments of three repeats. f , IP of IPMK with wild-type and mutant p53 from HCT116 and Cal33 cells respectively treated with 30 µM cisplatin or control vehicle for 24 h. Representative data of three independent experiments are shown. g , IP of PTEN with wild-type and mutant p53 from A549 and MDA-MB-231 cells respectively treated with 30 µM cisplatin or control vehicle for 24 h. Representative data of three independent experiments are shown. h-i , Quantification of the nuclear PLA foci of p53-PI4,5P 2 /PI3,4,5P 3 in MDA-MB-231 cells 48 h after transient transfection with control siRNAs or siRNAs against PIPKIα, IPMK, and PTEN. N=30 cells from representative experiments of three repeats. See PLA images in Extended Data Fig. 5k and knockdown validation by WB in Extended Data Fig. 5l. j , Schematic illustration of p53 in a complex with PI4,5P 2 and PI3,4,5P 3 downstream of PIPKIα and their interconversion by IPMK and PTEN. For all panels, data are represented as mean ±SD, p < 0.01 = **, t test. Scale bar: 5 µm.
    Figure Legend Snippet: a , Fluorescent IP-WB detects on-site PI4,5P 2 and PI3,4,5P 3 association with epitope-tagged p53 downstream of PIPKIα. FLAG-tagged mutant (R175H) or wild-type p53 were transient-transfected into HEK293FT cells for 24 h. Then the cells were treated with 50 µM PIPKIα inhibitor ISA-2011B (ISA) for 24 h. The ectopically-expressed p53 was IP-ed with FLAG antibody and analyzed by WB using fluorescent antibodies detecting PI4,5P 2 , PI3,4,5P 3 , and p53 simultaneously. N=3. b , Fluorescent IP-WB detects on-site PI4,5P 2 and PI3,4,5P 3 association with endogenous mutant p53 and stress-induced wild-type p53 in a panel of cancer cells. A549 cells expressing wild-type p53 were treated with 30 µM cisplatin or control vehicle for 24 h before being processed for IP against p53. BT-549, GILM2, HS578T, MDA-MB-231, and MDA-MB-468 cells expressing mutant p53 were directly processed for IP against mutant p53. The on-site PI4,5P 2 and PI3,4,5P 3 association with p53 was analyzed simultaneously by WB using fluorescent antibodies. N=3. c , Fluorescent IP-WB detects stress-induced PI3,4,5P 3 association with endogenous mutant p53 downstream of PIPKIα but independent from class I PI3Ks. MDA-MB-231 cells were treated with control vehicle or 30 µM cisplatin with or without the presence of the α-specific PI3K inhibitor alpelisib (10 µM), the pan-PI3K inhibitor buparlisib (2 µM), or the PIPKIα inhibitor ISA-2011B (50 µM) for 24 h. Then the cells were processed for IP against p53 and analyzed by WB using fluorescent antibodies detecting PI3,4,5P 3 and p53 simultaneously. N=3. d-e , PLA of p53-PI4,5P 2 /PI3,4,5P 3 /IPMK/PTEN overlaid with nuclear envelope marker Lamin A/C in MDA-MB-231 cells treated with control vehicle or 30 µM cisplatin for 24 h. The nuclear PLA foci were quantified ( e ). N=30 cells from representative experiments of three repeats. f , IP of IPMK with wild-type and mutant p53 from HCT116 and Cal33 cells respectively treated with 30 µM cisplatin or control vehicle for 24 h. Representative data of three independent experiments are shown. g , IP of PTEN with wild-type and mutant p53 from A549 and MDA-MB-231 cells respectively treated with 30 µM cisplatin or control vehicle for 24 h. Representative data of three independent experiments are shown. h-i , Quantification of the nuclear PLA foci of p53-PI4,5P 2 /PI3,4,5P 3 in MDA-MB-231 cells 48 h after transient transfection with control siRNAs or siRNAs against PIPKIα, IPMK, and PTEN. N=30 cells from representative experiments of three repeats. See PLA images in Extended Data Fig. 5k and knockdown validation by WB in Extended Data Fig. 5l. j , Schematic illustration of p53 in a complex with PI4,5P 2 and PI3,4,5P 3 downstream of PIPKIα and their interconversion by IPMK and PTEN. For all panels, data are represented as mean ±SD, p < 0.01 = **, t test. Scale bar: 5 µm.

    Techniques Used: Mutagenesis, Transfection, Expressing, Marker

    a-b , Triple fluorescent IP-WB detects stress-induced Akt association with the p53-PI3,4,5P 3 complex. MDA-MB-231 cells were treated with 30 µM cisplatin or control vehicle for 24 before being processed for IP against p53 and analyzed by WB using fluorescent antibodies detecting Akt, PI3,4,5P 3, and p53 simultaneously. The p53 associated PI3,4,5P 3 and Akt were quantified ( b ). N=3. c , MDA-MB-231 cells were transfected with control siRNAs or siRNAs against p53, PIPKIα, IPMK, and PTEN. 24 h later, cells were treated with 30 µM cisplatin or control vehicle for 24 h before being processed for IP against Akt. The Akt-associated p53 and the input of whole cell lysates were analyzed by WB. Representative images of three independent experiments are shown. d-e , IF staining of p53 and pAkt S473 in MDA-MB-231 cells 48 h after transient transfection with control siRNAs or siRNAs against p53, PIPKIα, IPMK, and PTEN. The nuclear pAkt S473 levels were quantified ( e ). N=30 cells from representative experiments of three repeats. f-g , MDA-MB-231 cells were transfected with control siRNAs or siRNAs against p53, PIPKIα, IPMK, and PTEN. 24 h later, cells were treated with 30 µM cisplatin or control vehicle for 24 h before processing for PLA between p53 and pAkt S473 . The nuclear PLA foci were quantified ( g ). N=30 cells from representative experiments of three repeats. h-i , IF staining of pAkt S473 in MDA-MB-231 cells treated with control vehicle or 30 µM cisplatin for 24 h with or without the presence of the PI3Kα inhibitor alpelisib (10 µM), the pan-PI3K inhibitor buparlisib (2 µM), or the PIPKIα inhibitor ISA-2011B (50 µM). The nuclear pAkt S473 levels were quantified ( i ). N=30 cells from representative experiments of three repeats. For all panels, data are represented as mean ±SD, p < 0.01 = **, t test. Scale bar: 5 µm.
    Figure Legend Snippet: a-b , Triple fluorescent IP-WB detects stress-induced Akt association with the p53-PI3,4,5P 3 complex. MDA-MB-231 cells were treated with 30 µM cisplatin or control vehicle for 24 before being processed for IP against p53 and analyzed by WB using fluorescent antibodies detecting Akt, PI3,4,5P 3, and p53 simultaneously. The p53 associated PI3,4,5P 3 and Akt were quantified ( b ). N=3. c , MDA-MB-231 cells were transfected with control siRNAs or siRNAs against p53, PIPKIα, IPMK, and PTEN. 24 h later, cells were treated with 30 µM cisplatin or control vehicle for 24 h before being processed for IP against Akt. The Akt-associated p53 and the input of whole cell lysates were analyzed by WB. Representative images of three independent experiments are shown. d-e , IF staining of p53 and pAkt S473 in MDA-MB-231 cells 48 h after transient transfection with control siRNAs or siRNAs against p53, PIPKIα, IPMK, and PTEN. The nuclear pAkt S473 levels were quantified ( e ). N=30 cells from representative experiments of three repeats. f-g , MDA-MB-231 cells were transfected with control siRNAs or siRNAs against p53, PIPKIα, IPMK, and PTEN. 24 h later, cells were treated with 30 µM cisplatin or control vehicle for 24 h before processing for PLA between p53 and pAkt S473 . The nuclear PLA foci were quantified ( g ). N=30 cells from representative experiments of three repeats. h-i , IF staining of pAkt S473 in MDA-MB-231 cells treated with control vehicle or 30 µM cisplatin for 24 h with or without the presence of the PI3Kα inhibitor alpelisib (10 µM), the pan-PI3K inhibitor buparlisib (2 µM), or the PIPKIα inhibitor ISA-2011B (50 µM). The nuclear pAkt S473 levels were quantified ( i ). N=30 cells from representative experiments of three repeats. For all panels, data are represented as mean ±SD, p < 0.01 = **, t test. Scale bar: 5 µm.

    Techniques Used: Transfection, Staining

    pip3 polypiposomes  (Echelon Biosciences)


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    Echelon Biosciences pip3 polypiposomes
    Pip3 Polypiposomes, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pip 3 polypiposomes y p039  (Echelon Biosciences)


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    Echelon Biosciences pip 3 polypiposomes y p039
    Pip 3 Polypiposomes Y P039, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pip 3 polypiposomes y p039  (Echelon Biosciences)


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    Echelon Biosciences pip 3 polypiposomes y p039
    Pip 3 Polypiposomes Y P039, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Echelon Biosciences y-p039
    Y P039, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Echelon Biosciences polypiposomes
    (A) Binding of tTG to recycling endosomes. The amounts of purified 125 I-tTG bound at 4°C to recycling endosomes immunoisolated from NIH3T3 cells lacking tTG were determined in a gamma counter with all measurements performed in triplicates. Shown are means ± SEM for three independent experiments. (B) The role of phosphoinositides in the tTG interaction with recycling endosomes. Binding of 50 nM purified tTG to recycling endosomes was tested in the presence of 100 µM free phosphoinositides. Bound tTG and endosomal Rab11A/Rab11B and transferrin receptor (TfR) were detected by immuno-blotting. The relative amounts of vesicle-bound tTG were quantified and compared to that in the sample without phosphoinositides in four independent experiments. Bars depict means ± SEM, *p<0.05. (C) The putative phospholipid-binding sequence and site within the fourth domain of tTG. (D) Comparative analysis of tTG binding to phosphoinositides. 125 I-tTG binding to synthetic liposomes <t>(PolyPIPosomes™)</t> containing indicated phiosphoinositides was determined in a gamma counter with all measurements performed in triplicates. Shown are means ± SEM for three independent experiments. (E) Association of tTG with phosphoinositides in cells. tTG was immunoprecipitated from extracts of WI-38 fibroblasts and the immune complexes were probed by immunoblotting for PI(3)P or PI(4)P. (F) The phospholipid-binding site in tTG is required for membrane targeting and externalization of the protein. K598A,K600A,R601A,K602A mutations (m-plbs) were generated within the phospholipid-binding site of tTG. NIH3T3 fibroblasts transfected with wild type tTG (wt) or its mutant deficient in phosphoinositide binding (m-plbs) were left untreated or treated with 10 mM neomycin for 24 h before induction of these proteins for 4 h. The contents of wt and m-plbs tTG in the recycling endosomes and cytosol were defined by immunoblotting ( left panels ). Cell surface and total levels of wt and m-plbs tTG were defined by surface biotinylation, isolation of surface protein fractions, and immunoblotting ( right panels ). The relative contents of wt and m-plbs tTG in the recycling endosomes and on the cell surface were quantified and compared to those for untreated cells expressing wt protein. Shown are representative of three independent experiments. Bars depict means ± SEM, **p<0.005. See also . (G) A proposed mechanism of unconventional tTG secretion. The de novo synthesized intracellular (cytoplasmic) tTG ( hexagons ) does not follow the classical, ER/Golgi-dependent secretion pathway, but is exported via a four-step unconventional secretion process mediated by recycling endosomes ( dotted arrows ). Solid arrows mark the major intracellular recycling route through the PNRC utilized by β1 integrins. See further comments in the text.
    Polypiposomes, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Echelon Biosciences pi3 4 5p 3 polypiposomes
    a , Fluorescent IP-WB detects on-site PI4,5P 2 and <t>PI3,4,5P</t> 3 association with epitope-tagged p53 downstream of PIPKIα. FLAG-tagged mutant (R175H) or wild-type p53 were transient-transfected into HEK293FT cells for 24 h. Then the cells were treated with 50 µM PIPKIα inhibitor ISA-2011B (ISA) for 24 h. The ectopically-expressed p53 was IP-ed with FLAG antibody and analyzed by WB using fluorescent antibodies detecting PI4,5P 2 , PI3,4,5P 3 , and p53 simultaneously. N=3. b , Fluorescent IP-WB detects on-site PI4,5P 2 and PI3,4,5P 3 association with endogenous mutant p53 and stress-induced wild-type p53 in a panel of cancer cells. A549 cells expressing wild-type p53 were treated with 30 µM cisplatin or control vehicle for 24 h before being processed for IP against p53. BT-549, GILM2, HS578T, MDA-MB-231, and MDA-MB-468 cells expressing mutant p53 were directly processed for IP against mutant p53. The on-site PI4,5P 2 and PI3,4,5P 3 association with p53 was analyzed simultaneously by WB using fluorescent antibodies. N=3. c , Fluorescent IP-WB detects stress-induced PI3,4,5P 3 association with endogenous mutant p53 downstream of PIPKIα but independent from class I PI3Ks. MDA-MB-231 cells were treated with control vehicle or 30 µM cisplatin with or without the presence of the α-specific PI3K inhibitor alpelisib (10 µM), the pan-PI3K inhibitor buparlisib (2 µM), or the PIPKIα inhibitor ISA-2011B (50 µM) for 24 h. Then the cells were processed for IP against p53 and analyzed by WB using fluorescent antibodies detecting PI3,4,5P 3 and p53 simultaneously. N=3. d-e , PLA of p53-PI4,5P 2 /PI3,4,5P 3 /IPMK/PTEN overlaid with nuclear envelope marker Lamin A/C in MDA-MB-231 cells treated with control vehicle or 30 µM cisplatin for 24 h. The nuclear PLA foci were quantified ( e ). N=30 cells from representative experiments of three repeats. f , IP of IPMK with wild-type and mutant p53 from HCT116 and Cal33 cells respectively treated with 30 µM cisplatin or control vehicle for 24 h. Representative data of three independent experiments are shown. g , IP of PTEN with wild-type and mutant p53 from A549 and MDA-MB-231 cells respectively treated with 30 µM cisplatin or control vehicle for 24 h. Representative data of three independent experiments are shown. h-i , Quantification of the nuclear PLA foci of p53-PI4,5P 2 /PI3,4,5P 3 in MDA-MB-231 cells 48 h after transient transfection with control siRNAs or siRNAs against PIPKIα, IPMK, and PTEN. N=30 cells from representative experiments of three repeats. See PLA images in Extended Data Fig. 5k and knockdown validation by WB in Extended Data Fig. 5l. j , Schematic illustration of p53 in a complex with PI4,5P 2 and PI3,4,5P 3 downstream of PIPKIα and their interconversion by IPMK and PTEN. For all panels, data are represented as mean ±SD, p < 0.01 = **, t test. Scale bar: 5 µm.
    Pi3 4 5p 3 Polypiposomes, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Echelon Biosciences pip3 polypiposomes
    a , Fluorescent IP-WB detects on-site PI4,5P 2 and <t>PI3,4,5P</t> 3 association with epitope-tagged p53 downstream of PIPKIα. FLAG-tagged mutant (R175H) or wild-type p53 were transient-transfected into HEK293FT cells for 24 h. Then the cells were treated with 50 µM PIPKIα inhibitor ISA-2011B (ISA) for 24 h. The ectopically-expressed p53 was IP-ed with FLAG antibody and analyzed by WB using fluorescent antibodies detecting PI4,5P 2 , PI3,4,5P 3 , and p53 simultaneously. N=3. b , Fluorescent IP-WB detects on-site PI4,5P 2 and PI3,4,5P 3 association with endogenous mutant p53 and stress-induced wild-type p53 in a panel of cancer cells. A549 cells expressing wild-type p53 were treated with 30 µM cisplatin or control vehicle for 24 h before being processed for IP against p53. BT-549, GILM2, HS578T, MDA-MB-231, and MDA-MB-468 cells expressing mutant p53 were directly processed for IP against mutant p53. The on-site PI4,5P 2 and PI3,4,5P 3 association with p53 was analyzed simultaneously by WB using fluorescent antibodies. N=3. c , Fluorescent IP-WB detects stress-induced PI3,4,5P 3 association with endogenous mutant p53 downstream of PIPKIα but independent from class I PI3Ks. MDA-MB-231 cells were treated with control vehicle or 30 µM cisplatin with or without the presence of the α-specific PI3K inhibitor alpelisib (10 µM), the pan-PI3K inhibitor buparlisib (2 µM), or the PIPKIα inhibitor ISA-2011B (50 µM) for 24 h. Then the cells were processed for IP against p53 and analyzed by WB using fluorescent antibodies detecting PI3,4,5P 3 and p53 simultaneously. N=3. d-e , PLA of p53-PI4,5P 2 /PI3,4,5P 3 /IPMK/PTEN overlaid with nuclear envelope marker Lamin A/C in MDA-MB-231 cells treated with control vehicle or 30 µM cisplatin for 24 h. The nuclear PLA foci were quantified ( e ). N=30 cells from representative experiments of three repeats. f , IP of IPMK with wild-type and mutant p53 from HCT116 and Cal33 cells respectively treated with 30 µM cisplatin or control vehicle for 24 h. Representative data of three independent experiments are shown. g , IP of PTEN with wild-type and mutant p53 from A549 and MDA-MB-231 cells respectively treated with 30 µM cisplatin or control vehicle for 24 h. Representative data of three independent experiments are shown. h-i , Quantification of the nuclear PLA foci of p53-PI4,5P 2 /PI3,4,5P 3 in MDA-MB-231 cells 48 h after transient transfection with control siRNAs or siRNAs against PIPKIα, IPMK, and PTEN. N=30 cells from representative experiments of three repeats. See PLA images in Extended Data Fig. 5k and knockdown validation by WB in Extended Data Fig. 5l. j , Schematic illustration of p53 in a complex with PI4,5P 2 and PI3,4,5P 3 downstream of PIPKIα and their interconversion by IPMK and PTEN. For all panels, data are represented as mean ±SD, p < 0.01 = **, t test. Scale bar: 5 µm.
    Pip3 Polypiposomes, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Echelon Biosciences pip 3 polypiposomes y p039
    a , Fluorescent IP-WB detects on-site PI4,5P 2 and <t>PI3,4,5P</t> 3 association with epitope-tagged p53 downstream of PIPKIα. FLAG-tagged mutant (R175H) or wild-type p53 were transient-transfected into HEK293FT cells for 24 h. Then the cells were treated with 50 µM PIPKIα inhibitor ISA-2011B (ISA) for 24 h. The ectopically-expressed p53 was IP-ed with FLAG antibody and analyzed by WB using fluorescent antibodies detecting PI4,5P 2 , PI3,4,5P 3 , and p53 simultaneously. N=3. b , Fluorescent IP-WB detects on-site PI4,5P 2 and PI3,4,5P 3 association with endogenous mutant p53 and stress-induced wild-type p53 in a panel of cancer cells. A549 cells expressing wild-type p53 were treated with 30 µM cisplatin or control vehicle for 24 h before being processed for IP against p53. BT-549, GILM2, HS578T, MDA-MB-231, and MDA-MB-468 cells expressing mutant p53 were directly processed for IP against mutant p53. The on-site PI4,5P 2 and PI3,4,5P 3 association with p53 was analyzed simultaneously by WB using fluorescent antibodies. N=3. c , Fluorescent IP-WB detects stress-induced PI3,4,5P 3 association with endogenous mutant p53 downstream of PIPKIα but independent from class I PI3Ks. MDA-MB-231 cells were treated with control vehicle or 30 µM cisplatin with or without the presence of the α-specific PI3K inhibitor alpelisib (10 µM), the pan-PI3K inhibitor buparlisib (2 µM), or the PIPKIα inhibitor ISA-2011B (50 µM) for 24 h. Then the cells were processed for IP against p53 and analyzed by WB using fluorescent antibodies detecting PI3,4,5P 3 and p53 simultaneously. N=3. d-e , PLA of p53-PI4,5P 2 /PI3,4,5P 3 /IPMK/PTEN overlaid with nuclear envelope marker Lamin A/C in MDA-MB-231 cells treated with control vehicle or 30 µM cisplatin for 24 h. The nuclear PLA foci were quantified ( e ). N=30 cells from representative experiments of three repeats. f , IP of IPMK with wild-type and mutant p53 from HCT116 and Cal33 cells respectively treated with 30 µM cisplatin or control vehicle for 24 h. Representative data of three independent experiments are shown. g , IP of PTEN with wild-type and mutant p53 from A549 and MDA-MB-231 cells respectively treated with 30 µM cisplatin or control vehicle for 24 h. Representative data of three independent experiments are shown. h-i , Quantification of the nuclear PLA foci of p53-PI4,5P 2 /PI3,4,5P 3 in MDA-MB-231 cells 48 h after transient transfection with control siRNAs or siRNAs against PIPKIα, IPMK, and PTEN. N=30 cells from representative experiments of three repeats. See PLA images in Extended Data Fig. 5k and knockdown validation by WB in Extended Data Fig. 5l. j , Schematic illustration of p53 in a complex with PI4,5P 2 and PI3,4,5P 3 downstream of PIPKIα and their interconversion by IPMK and PTEN. For all panels, data are represented as mean ±SD, p < 0.01 = **, t test. Scale bar: 5 µm.
    Pip 3 Polypiposomes Y P039, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    (A) Binding of tTG to recycling endosomes. The amounts of purified 125 I-tTG bound at 4°C to recycling endosomes immunoisolated from NIH3T3 cells lacking tTG were determined in a gamma counter with all measurements performed in triplicates. Shown are means ± SEM for three independent experiments. (B) The role of phosphoinositides in the tTG interaction with recycling endosomes. Binding of 50 nM purified tTG to recycling endosomes was tested in the presence of 100 µM free phosphoinositides. Bound tTG and endosomal Rab11A/Rab11B and transferrin receptor (TfR) were detected by immuno-blotting. The relative amounts of vesicle-bound tTG were quantified and compared to that in the sample without phosphoinositides in four independent experiments. Bars depict means ± SEM, *p<0.05. (C) The putative phospholipid-binding sequence and site within the fourth domain of tTG. (D) Comparative analysis of tTG binding to phosphoinositides. 125 I-tTG binding to synthetic liposomes (PolyPIPosomes™) containing indicated phiosphoinositides was determined in a gamma counter with all measurements performed in triplicates. Shown are means ± SEM for three independent experiments. (E) Association of tTG with phosphoinositides in cells. tTG was immunoprecipitated from extracts of WI-38 fibroblasts and the immune complexes were probed by immunoblotting for PI(3)P or PI(4)P. (F) The phospholipid-binding site in tTG is required for membrane targeting and externalization of the protein. K598A,K600A,R601A,K602A mutations (m-plbs) were generated within the phospholipid-binding site of tTG. NIH3T3 fibroblasts transfected with wild type tTG (wt) or its mutant deficient in phosphoinositide binding (m-plbs) were left untreated or treated with 10 mM neomycin for 24 h before induction of these proteins for 4 h. The contents of wt and m-plbs tTG in the recycling endosomes and cytosol were defined by immunoblotting ( left panels ). Cell surface and total levels of wt and m-plbs tTG were defined by surface biotinylation, isolation of surface protein fractions, and immunoblotting ( right panels ). The relative contents of wt and m-plbs tTG in the recycling endosomes and on the cell surface were quantified and compared to those for untreated cells expressing wt protein. Shown are representative of three independent experiments. Bars depict means ± SEM, **p<0.005. See also . (G) A proposed mechanism of unconventional tTG secretion. The de novo synthesized intracellular (cytoplasmic) tTG ( hexagons ) does not follow the classical, ER/Golgi-dependent secretion pathway, but is exported via a four-step unconventional secretion process mediated by recycling endosomes ( dotted arrows ). Solid arrows mark the major intracellular recycling route through the PNRC utilized by β1 integrins. See further comments in the text.

    Journal: PLoS ONE

    Article Title: Unconventional Secretion of Tissue Transglutaminase Involves Phospholipid-Dependent Delivery into Recycling Endosomes

    doi: 10.1371/journal.pone.0019414

    Figure Lengend Snippet: (A) Binding of tTG to recycling endosomes. The amounts of purified 125 I-tTG bound at 4°C to recycling endosomes immunoisolated from NIH3T3 cells lacking tTG were determined in a gamma counter with all measurements performed in triplicates. Shown are means ± SEM for three independent experiments. (B) The role of phosphoinositides in the tTG interaction with recycling endosomes. Binding of 50 nM purified tTG to recycling endosomes was tested in the presence of 100 µM free phosphoinositides. Bound tTG and endosomal Rab11A/Rab11B and transferrin receptor (TfR) were detected by immuno-blotting. The relative amounts of vesicle-bound tTG were quantified and compared to that in the sample without phosphoinositides in four independent experiments. Bars depict means ± SEM, *p<0.05. (C) The putative phospholipid-binding sequence and site within the fourth domain of tTG. (D) Comparative analysis of tTG binding to phosphoinositides. 125 I-tTG binding to synthetic liposomes (PolyPIPosomes™) containing indicated phiosphoinositides was determined in a gamma counter with all measurements performed in triplicates. Shown are means ± SEM for three independent experiments. (E) Association of tTG with phosphoinositides in cells. tTG was immunoprecipitated from extracts of WI-38 fibroblasts and the immune complexes were probed by immunoblotting for PI(3)P or PI(4)P. (F) The phospholipid-binding site in tTG is required for membrane targeting and externalization of the protein. K598A,K600A,R601A,K602A mutations (m-plbs) were generated within the phospholipid-binding site of tTG. NIH3T3 fibroblasts transfected with wild type tTG (wt) or its mutant deficient in phosphoinositide binding (m-plbs) were left untreated or treated with 10 mM neomycin for 24 h before induction of these proteins for 4 h. The contents of wt and m-plbs tTG in the recycling endosomes and cytosol were defined by immunoblotting ( left panels ). Cell surface and total levels of wt and m-plbs tTG were defined by surface biotinylation, isolation of surface protein fractions, and immunoblotting ( right panels ). The relative contents of wt and m-plbs tTG in the recycling endosomes and on the cell surface were quantified and compared to those for untreated cells expressing wt protein. Shown are representative of three independent experiments. Bars depict means ± SEM, **p<0.005. See also . (G) A proposed mechanism of unconventional tTG secretion. The de novo synthesized intracellular (cytoplasmic) tTG ( hexagons ) does not follow the classical, ER/Golgi-dependent secretion pathway, but is exported via a four-step unconventional secretion process mediated by recycling endosomes ( dotted arrows ). Solid arrows mark the major intracellular recycling route through the PNRC utilized by β1 integrins. See further comments in the text.

    Article Snippet: Membrane lipid strips (#P6002); PIP strips (#P6001), PolyPIPosomes (#YP000; #YP003; #YP004; #YP005; #YP034; #YP035; #YP045; #YP039), and purified synthetic phosphoinositides were from Echelon Biosciences.

    Techniques: Binding Assay, Purification, Sequencing, Immunoprecipitation, Western Blot, Generated, Transfection, Mutagenesis, Isolation, Expressing, Synthesized

    a , Fluorescent IP-WB detects on-site PI4,5P 2 and PI3,4,5P 3 association with epitope-tagged p53 downstream of PIPKIα. FLAG-tagged mutant (R175H) or wild-type p53 were transient-transfected into HEK293FT cells for 24 h. Then the cells were treated with 50 µM PIPKIα inhibitor ISA-2011B (ISA) for 24 h. The ectopically-expressed p53 was IP-ed with FLAG antibody and analyzed by WB using fluorescent antibodies detecting PI4,5P 2 , PI3,4,5P 3 , and p53 simultaneously. N=3. b , Fluorescent IP-WB detects on-site PI4,5P 2 and PI3,4,5P 3 association with endogenous mutant p53 and stress-induced wild-type p53 in a panel of cancer cells. A549 cells expressing wild-type p53 were treated with 30 µM cisplatin or control vehicle for 24 h before being processed for IP against p53. BT-549, GILM2, HS578T, MDA-MB-231, and MDA-MB-468 cells expressing mutant p53 were directly processed for IP against mutant p53. The on-site PI4,5P 2 and PI3,4,5P 3 association with p53 was analyzed simultaneously by WB using fluorescent antibodies. N=3. c , Fluorescent IP-WB detects stress-induced PI3,4,5P 3 association with endogenous mutant p53 downstream of PIPKIα but independent from class I PI3Ks. MDA-MB-231 cells were treated with control vehicle or 30 µM cisplatin with or without the presence of the α-specific PI3K inhibitor alpelisib (10 µM), the pan-PI3K inhibitor buparlisib (2 µM), or the PIPKIα inhibitor ISA-2011B (50 µM) for 24 h. Then the cells were processed for IP against p53 and analyzed by WB using fluorescent antibodies detecting PI3,4,5P 3 and p53 simultaneously. N=3. d-e , PLA of p53-PI4,5P 2 /PI3,4,5P 3 /IPMK/PTEN overlaid with nuclear envelope marker Lamin A/C in MDA-MB-231 cells treated with control vehicle or 30 µM cisplatin for 24 h. The nuclear PLA foci were quantified ( e ). N=30 cells from representative experiments of three repeats. f , IP of IPMK with wild-type and mutant p53 from HCT116 and Cal33 cells respectively treated with 30 µM cisplatin or control vehicle for 24 h. Representative data of three independent experiments are shown. g , IP of PTEN with wild-type and mutant p53 from A549 and MDA-MB-231 cells respectively treated with 30 µM cisplatin or control vehicle for 24 h. Representative data of three independent experiments are shown. h-i , Quantification of the nuclear PLA foci of p53-PI4,5P 2 /PI3,4,5P 3 in MDA-MB-231 cells 48 h after transient transfection with control siRNAs or siRNAs against PIPKIα, IPMK, and PTEN. N=30 cells from representative experiments of three repeats. See PLA images in Extended Data Fig. 5k and knockdown validation by WB in Extended Data Fig. 5l. j , Schematic illustration of p53 in a complex with PI4,5P 2 and PI3,4,5P 3 downstream of PIPKIα and their interconversion by IPMK and PTEN. For all panels, data are represented as mean ±SD, p < 0.01 = **, t test. Scale bar: 5 µm.

    Journal: bioRxiv

    Article Title: A p53-Phosphoinositide Signalosome Regulates Nuclear Akt Activation

    doi: 10.1101/2021.09.17.460854

    Figure Lengend Snippet: a , Fluorescent IP-WB detects on-site PI4,5P 2 and PI3,4,5P 3 association with epitope-tagged p53 downstream of PIPKIα. FLAG-tagged mutant (R175H) or wild-type p53 were transient-transfected into HEK293FT cells for 24 h. Then the cells were treated with 50 µM PIPKIα inhibitor ISA-2011B (ISA) for 24 h. The ectopically-expressed p53 was IP-ed with FLAG antibody and analyzed by WB using fluorescent antibodies detecting PI4,5P 2 , PI3,4,5P 3 , and p53 simultaneously. N=3. b , Fluorescent IP-WB detects on-site PI4,5P 2 and PI3,4,5P 3 association with endogenous mutant p53 and stress-induced wild-type p53 in a panel of cancer cells. A549 cells expressing wild-type p53 were treated with 30 µM cisplatin or control vehicle for 24 h before being processed for IP against p53. BT-549, GILM2, HS578T, MDA-MB-231, and MDA-MB-468 cells expressing mutant p53 were directly processed for IP against mutant p53. The on-site PI4,5P 2 and PI3,4,5P 3 association with p53 was analyzed simultaneously by WB using fluorescent antibodies. N=3. c , Fluorescent IP-WB detects stress-induced PI3,4,5P 3 association with endogenous mutant p53 downstream of PIPKIα but independent from class I PI3Ks. MDA-MB-231 cells were treated with control vehicle or 30 µM cisplatin with or without the presence of the α-specific PI3K inhibitor alpelisib (10 µM), the pan-PI3K inhibitor buparlisib (2 µM), or the PIPKIα inhibitor ISA-2011B (50 µM) for 24 h. Then the cells were processed for IP against p53 and analyzed by WB using fluorescent antibodies detecting PI3,4,5P 3 and p53 simultaneously. N=3. d-e , PLA of p53-PI4,5P 2 /PI3,4,5P 3 /IPMK/PTEN overlaid with nuclear envelope marker Lamin A/C in MDA-MB-231 cells treated with control vehicle or 30 µM cisplatin for 24 h. The nuclear PLA foci were quantified ( e ). N=30 cells from representative experiments of three repeats. f , IP of IPMK with wild-type and mutant p53 from HCT116 and Cal33 cells respectively treated with 30 µM cisplatin or control vehicle for 24 h. Representative data of three independent experiments are shown. g , IP of PTEN with wild-type and mutant p53 from A549 and MDA-MB-231 cells respectively treated with 30 µM cisplatin or control vehicle for 24 h. Representative data of three independent experiments are shown. h-i , Quantification of the nuclear PLA foci of p53-PI4,5P 2 /PI3,4,5P 3 in MDA-MB-231 cells 48 h after transient transfection with control siRNAs or siRNAs against PIPKIα, IPMK, and PTEN. N=30 cells from representative experiments of three repeats. See PLA images in Extended Data Fig. 5k and knockdown validation by WB in Extended Data Fig. 5l. j , Schematic illustration of p53 in a complex with PI4,5P 2 and PI3,4,5P 3 downstream of PIPKIα and their interconversion by IPMK and PTEN. For all panels, data are represented as mean ±SD, p < 0.01 = **, t test. Scale bar: 5 µm.

    Article Snippet: The PI-PolyPIPosomes for MST were purchased from Echelon Biosciences, including PI PolyPIPosomes (#Y-P000), PI4,5P 2 PolyPIPosomes (#Y-P045), and PI3,4,5P 3 PolyPIPosomes (#Y-P039).

    Techniques: Mutagenesis, Transfection, Expressing, Marker

    a-b , Triple fluorescent IP-WB detects stress-induced Akt association with the p53-PI3,4,5P 3 complex. MDA-MB-231 cells were treated with 30 µM cisplatin or control vehicle for 24 before being processed for IP against p53 and analyzed by WB using fluorescent antibodies detecting Akt, PI3,4,5P 3, and p53 simultaneously. The p53 associated PI3,4,5P 3 and Akt were quantified ( b ). N=3. c , MDA-MB-231 cells were transfected with control siRNAs or siRNAs against p53, PIPKIα, IPMK, and PTEN. 24 h later, cells were treated with 30 µM cisplatin or control vehicle for 24 h before being processed for IP against Akt. The Akt-associated p53 and the input of whole cell lysates were analyzed by WB. Representative images of three independent experiments are shown. d-e , IF staining of p53 and pAkt S473 in MDA-MB-231 cells 48 h after transient transfection with control siRNAs or siRNAs against p53, PIPKIα, IPMK, and PTEN. The nuclear pAkt S473 levels were quantified ( e ). N=30 cells from representative experiments of three repeats. f-g , MDA-MB-231 cells were transfected with control siRNAs or siRNAs against p53, PIPKIα, IPMK, and PTEN. 24 h later, cells were treated with 30 µM cisplatin or control vehicle for 24 h before processing for PLA between p53 and pAkt S473 . The nuclear PLA foci were quantified ( g ). N=30 cells from representative experiments of three repeats. h-i , IF staining of pAkt S473 in MDA-MB-231 cells treated with control vehicle or 30 µM cisplatin for 24 h with or without the presence of the PI3Kα inhibitor alpelisib (10 µM), the pan-PI3K inhibitor buparlisib (2 µM), or the PIPKIα inhibitor ISA-2011B (50 µM). The nuclear pAkt S473 levels were quantified ( i ). N=30 cells from representative experiments of three repeats. For all panels, data are represented as mean ±SD, p < 0.01 = **, t test. Scale bar: 5 µm.

    Journal: bioRxiv

    Article Title: A p53-Phosphoinositide Signalosome Regulates Nuclear Akt Activation

    doi: 10.1101/2021.09.17.460854

    Figure Lengend Snippet: a-b , Triple fluorescent IP-WB detects stress-induced Akt association with the p53-PI3,4,5P 3 complex. MDA-MB-231 cells were treated with 30 µM cisplatin or control vehicle for 24 before being processed for IP against p53 and analyzed by WB using fluorescent antibodies detecting Akt, PI3,4,5P 3, and p53 simultaneously. The p53 associated PI3,4,5P 3 and Akt were quantified ( b ). N=3. c , MDA-MB-231 cells were transfected with control siRNAs or siRNAs against p53, PIPKIα, IPMK, and PTEN. 24 h later, cells were treated with 30 µM cisplatin or control vehicle for 24 h before being processed for IP against Akt. The Akt-associated p53 and the input of whole cell lysates were analyzed by WB. Representative images of three independent experiments are shown. d-e , IF staining of p53 and pAkt S473 in MDA-MB-231 cells 48 h after transient transfection with control siRNAs or siRNAs against p53, PIPKIα, IPMK, and PTEN. The nuclear pAkt S473 levels were quantified ( e ). N=30 cells from representative experiments of three repeats. f-g , MDA-MB-231 cells were transfected with control siRNAs or siRNAs against p53, PIPKIα, IPMK, and PTEN. 24 h later, cells were treated with 30 µM cisplatin or control vehicle for 24 h before processing for PLA between p53 and pAkt S473 . The nuclear PLA foci were quantified ( g ). N=30 cells from representative experiments of three repeats. h-i , IF staining of pAkt S473 in MDA-MB-231 cells treated with control vehicle or 30 µM cisplatin for 24 h with or without the presence of the PI3Kα inhibitor alpelisib (10 µM), the pan-PI3K inhibitor buparlisib (2 µM), or the PIPKIα inhibitor ISA-2011B (50 µM). The nuclear pAkt S473 levels were quantified ( i ). N=30 cells from representative experiments of three repeats. For all panels, data are represented as mean ±SD, p < 0.01 = **, t test. Scale bar: 5 µm.

    Article Snippet: The PI-PolyPIPosomes for MST were purchased from Echelon Biosciences, including PI PolyPIPosomes (#Y-P000), PI4,5P 2 PolyPIPosomes (#Y-P045), and PI3,4,5P 3 PolyPIPosomes (#Y-P039).

    Techniques: Transfection, Staining