pip3  (Echelon Biosciences)


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    Structured Review

    Echelon Biosciences pip3
    Structural model for the differential effect of T280 phosphorylation on <t>PIP3</t> vs. PI4P binding to the Grp1 PH domain. The phosphoinositide-binding site in the crystal structure of the Grp1 PH domain bound to the PIP3 head group (PDB ID 1FGY) is compared with a hypothetical composite model for the PI4P head group. The PI4P head group was acquired from the crystal structure of the ARNO PH domain bound to the PIP2 head group (PDB ID 1U29) after alignment of the PH domains and deletion of the 5-phosphate. Magenta dashes represent hydrogen bonds between the T280 side chain hydroxyl group and the 1-phosphate of the PIP3 head group that are not observed for the PIP2 head group in the ARNO complex.
    Pip3, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pip3/product/Echelon Biosciences
    Average 94 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    pip3 - by Bioz Stars, 2022-08
    94/100 stars

    Images

    1) Product Images from "Coordination of Grp1 recruitment mechanisms by its phosphorylation"

    Article Title: Coordination of Grp1 recruitment mechanisms by its phosphorylation

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E20-03-0173

    Structural model for the differential effect of T280 phosphorylation on PIP3 vs. PI4P binding to the Grp1 PH domain. The phosphoinositide-binding site in the crystal structure of the Grp1 PH domain bound to the PIP3 head group (PDB ID 1FGY) is compared with a hypothetical composite model for the PI4P head group. The PI4P head group was acquired from the crystal structure of the ARNO PH domain bound to the PIP2 head group (PDB ID 1U29) after alignment of the PH domains and deletion of the 5-phosphate. Magenta dashes represent hydrogen bonds between the T280 side chain hydroxyl group and the 1-phosphate of the PIP3 head group that are not observed for the PIP2 head group in the ARNO complex.
    Figure Legend Snippet: Structural model for the differential effect of T280 phosphorylation on PIP3 vs. PI4P binding to the Grp1 PH domain. The phosphoinositide-binding site in the crystal structure of the Grp1 PH domain bound to the PIP3 head group (PDB ID 1FGY) is compared with a hypothetical composite model for the PI4P head group. The PI4P head group was acquired from the crystal structure of the ARNO PH domain bound to the PIP2 head group (PDB ID 1U29) after alignment of the PH domains and deletion of the 5-phosphate. Magenta dashes represent hydrogen bonds between the T280 side chain hydroxyl group and the 1-phosphate of the PIP3 head group that are not observed for the PIP2 head group in the ARNO complex.

    Techniques Used: Binding Assay

    2) Product Images from "Externalized phosphatidylinositides on apoptotic cells are eat-me signals recognized by CD14"

    Article Title: Externalized phosphatidylinositides on apoptotic cells are eat-me signals recognized by CD14

    Journal: Cell Death and Differentiation

    doi: 10.1038/s41418-022-00931-2

    In vitro induction of exofacial PIPs. A , B Flow cytometric analyses of Jurkat cells treated 0–16 h with 10 μM camptothecin. A Representative flow cytometric plots (left) of TO-PRO-3 (TP-3) and propidium iodide (PI)-stained cells showing R1 (TP-3 + PI − ), R2 (TP-3 + PI + ), R3 (TP-3 − PI + ), and R4 (TP-3 − PI − ) subsets. Histograms (right) show the binding levels of Annexin V, AKT PHD, CD14, or PIP 3 antibody in R1 (dark gray), R2 (red), and R4 (light grey) subsets. B Percentage of Annexin V, AKT PHD, CD14, and PI(3,4,5)P 3 in R1 (upper low) and R2 (lower low) cells. C , D Flow cytometric analyses of Jurkat cells treated 16 h with 10 μM camptothecin. C Representative flow cytometric plots (left) and binding percentages (right) of an AKT PHD and CD14 combination. D Representative flow cytometric plots (left) and binding percentages (right) of an Annexin V and AKT PHD combination. E Representative flow cytometric plots (left) and binding percentages (right) of an Annexin V and CD14 combination. F CHO cells treated 6 h with 10 μM camptothecin and incubated with Annexin V and AKT PHD were detected with primary antibodies and Texas Red- or FITC-labeled secondary antibodies, respectively. See also Supplementary Fig. 8 and Supplementary Movie 2 . All comparisons were performed by one-way ANOVA with Tukey’s post-hoc multiple comparison test. All data are presented as the mean ± SD for each group ( n = 6 per group unless noted otherwise). * p
    Figure Legend Snippet: In vitro induction of exofacial PIPs. A , B Flow cytometric analyses of Jurkat cells treated 0–16 h with 10 μM camptothecin. A Representative flow cytometric plots (left) of TO-PRO-3 (TP-3) and propidium iodide (PI)-stained cells showing R1 (TP-3 + PI − ), R2 (TP-3 + PI + ), R3 (TP-3 − PI + ), and R4 (TP-3 − PI − ) subsets. Histograms (right) show the binding levels of Annexin V, AKT PHD, CD14, or PIP 3 antibody in R1 (dark gray), R2 (red), and R4 (light grey) subsets. B Percentage of Annexin V, AKT PHD, CD14, and PI(3,4,5)P 3 in R1 (upper low) and R2 (lower low) cells. C , D Flow cytometric analyses of Jurkat cells treated 16 h with 10 μM camptothecin. C Representative flow cytometric plots (left) and binding percentages (right) of an AKT PHD and CD14 combination. D Representative flow cytometric plots (left) and binding percentages (right) of an Annexin V and AKT PHD combination. E Representative flow cytometric plots (left) and binding percentages (right) of an Annexin V and CD14 combination. F CHO cells treated 6 h with 10 μM camptothecin and incubated with Annexin V and AKT PHD were detected with primary antibodies and Texas Red- or FITC-labeled secondary antibodies, respectively. See also Supplementary Fig. 8 and Supplementary Movie 2 . All comparisons were performed by one-way ANOVA with Tukey’s post-hoc multiple comparison test. All data are presented as the mean ± SD for each group ( n = 6 per group unless noted otherwise). * p

    Techniques Used: In Vitro, Staining, Binding Assay, Incubation, Labeling

    PIP externalization and its role in phagocytosis. A Representative images of apoptotic cell detection by recombinant WT CD14 or mutant R4A CD14 protein. After inducing apoptosis with an anti-FAS antibody (100 ng/ml), HeLa cells incubated with recombinant WT or R4A CD14 were visualized using an anti-CD14 primary antibody and an FITC-labeled secondary antibody (scale bars, 10 μm). B Representative images of TPM phagocytosis of camptothecin-treated, pHrodo-labeled Jurkat cells using an antibody or recombinant proteins to mask externalized PS or PIPs. TPM phagocytosis of camptothecin-treated, pHrodo-labeled Jurkat cells was inhibited by an anti-PI(3,4,5)P 3 antibody, AKT PHD, Annexin V, and CD14 (scale bars, 50 μm). The red color represents engulfed apoptotic cells labeled with pHrodo dyes. C TPM phagocytosis of camptothecin-treated, pHrodo-labeled Jurkat cells was quantified in 8–10 low-power fields (mean ± SD, n = 5–10). D Representative flow cytometric plots (left) and percentages (right) of F4/80 + TPM phagocytosis of pHrodo-labeled thymocytes isolated from irradiated WT or Cd14 −/− mice group ( n = 6 per group). Thymocytes from irradiated mice were labeled with pHrodo for 30 min prior to adding TPMs from WT or Cd14 −/− mice for 1 hr (mean ± SD, n = 6). E Representative flow cytometry plots (upper) and MFI values (lower) of in vivo phagocytosis of pHrodo-labeled apoptotic thymocytes by F4/80+ mouse peritoneal macrophages of WT or Cd14 −/− mice group ( n = 3 per group). Thymocytes from irradiated mice were labeled with pHrodo for 30 min prior to injecting the peritoneal cavity of WT or Cd14 −/− mice for 15 min. F Representative fluorescent images (left) and intensities (right) of engulfed synthetic Bodipy-labeled PI(3,4,5)P 3 in TPMs from WT or Cd14 −/− mice. Engulfed Bodipy-labeled PIP 3 was visualized by confocal microscopy and quantified by fluorescence intensity using NIH ImageJ software (mean ± SD, n = 10) after incubating the TPMs with 1 μg of Bodipy-labeled PI(3,4,5)P 3 per well for 30 min (scale bars, 10 μm). G Representative flow cytometric plots (left) and MFI values (right) of engulfed synthetic Bodipy-labeled PI(3,4,5)P 3 in PI - /F4/80 + TPMs from WT or Cd14 −/− mice (mean ± SD, n = 6). All comparisons were performed by one-way ANOVA with Tukey’s post-hoc multiple comparison test Fig. 3A) or unpaired student t tests (Fig. 4D–F ). All data are presented as the mean ± SD for each group. * p
    Figure Legend Snippet: PIP externalization and its role in phagocytosis. A Representative images of apoptotic cell detection by recombinant WT CD14 or mutant R4A CD14 protein. After inducing apoptosis with an anti-FAS antibody (100 ng/ml), HeLa cells incubated with recombinant WT or R4A CD14 were visualized using an anti-CD14 primary antibody and an FITC-labeled secondary antibody (scale bars, 10 μm). B Representative images of TPM phagocytosis of camptothecin-treated, pHrodo-labeled Jurkat cells using an antibody or recombinant proteins to mask externalized PS or PIPs. TPM phagocytosis of camptothecin-treated, pHrodo-labeled Jurkat cells was inhibited by an anti-PI(3,4,5)P 3 antibody, AKT PHD, Annexin V, and CD14 (scale bars, 50 μm). The red color represents engulfed apoptotic cells labeled with pHrodo dyes. C TPM phagocytosis of camptothecin-treated, pHrodo-labeled Jurkat cells was quantified in 8–10 low-power fields (mean ± SD, n = 5–10). D Representative flow cytometric plots (left) and percentages (right) of F4/80 + TPM phagocytosis of pHrodo-labeled thymocytes isolated from irradiated WT or Cd14 −/− mice group ( n = 6 per group). Thymocytes from irradiated mice were labeled with pHrodo for 30 min prior to adding TPMs from WT or Cd14 −/− mice for 1 hr (mean ± SD, n = 6). E Representative flow cytometry plots (upper) and MFI values (lower) of in vivo phagocytosis of pHrodo-labeled apoptotic thymocytes by F4/80+ mouse peritoneal macrophages of WT or Cd14 −/− mice group ( n = 3 per group). Thymocytes from irradiated mice were labeled with pHrodo for 30 min prior to injecting the peritoneal cavity of WT or Cd14 −/− mice for 15 min. F Representative fluorescent images (left) and intensities (right) of engulfed synthetic Bodipy-labeled PI(3,4,5)P 3 in TPMs from WT or Cd14 −/− mice. Engulfed Bodipy-labeled PIP 3 was visualized by confocal microscopy and quantified by fluorescence intensity using NIH ImageJ software (mean ± SD, n = 10) after incubating the TPMs with 1 μg of Bodipy-labeled PI(3,4,5)P 3 per well for 30 min (scale bars, 10 μm). G Representative flow cytometric plots (left) and MFI values (right) of engulfed synthetic Bodipy-labeled PI(3,4,5)P 3 in PI - /F4/80 + TPMs from WT or Cd14 −/− mice (mean ± SD, n = 6). All comparisons were performed by one-way ANOVA with Tukey’s post-hoc multiple comparison test Fig. 3A) or unpaired student t tests (Fig. 4D–F ). All data are presented as the mean ± SD for each group. * p

    Techniques Used: Recombinant, Mutagenesis, Incubation, Labeling, Isolation, Irradiation, Mouse Assay, Flow Cytometry, In Vivo, Confocal Microscopy, Fluorescence, Software

    The role of CD14 + macrophages in IC-IP 6 uptake and PIP recognition. Representative liver sections (left) and quantitative data (right) from mice treated with or without intravenous IC-IP 6 ( n = 6 per group). The liver sections were stained with Prussian blue (PB) for iron and anti-F4/80 antibodies for macrophages/Kupffer cells (scale bars, 20 μm). F4/80+ PB+ cells were quantified using ImageJ software. B , C LPS induction of IC-IP 6 uptake by TPMs, determined by PB-stained cells (scale bars, 20 μm) ( B ), or ( C ) by iron staining with 2,4,6-tri-(2-pyridyl)-5-triazine (TPTZ) in cell lysates ( n = 8). Each point represents the mean of three experimental replicates for each IC-IP 6 concentration. D Proteins isolated from RAW264.7 cell lysates by IC-IP 6 precipitation were eluted with NaCl, separated by SDS-PAGE, and visualized using Coomassie Brilliant Blue (black arrowhead = CD14). CD14 identity was confirmed by western blotting. E , F CD14 binding to immobilized phospholipids (100 pmol each) ( E ), and varying concentrations of phosphatidylinositol phosphates (PIP strips or PIP arrays, Echelon Biosciences) ( F ). G Representative flow cytometry analyses of the binding of CD14, AKT PHD, or anti-PIP 3 antibody to the indicated phospholipids. Silica particles loaded with specific combinations of phospholipids (Echelon Bioscience) were treated with anti-PI(3,4,5)P 3 antibody or His-tagged AKT PHD, or CD14 protein. Samples with His-tagged proteins were treated with primary anti-His antibody, and all were visualized using an Alexa-647-labeled secondary antibody. The blue dotted lines indicate the peaks of the isotype controls. H ITC results for IP 6 titration into 30 μM CD14; the K d value was determined by curve fitting the raw data ( n = 2; MicroCal). I Modeled PI(3,4,5)P 3 -CD14 structure, generated with AutoDock PyRx and coordinates for IP 6 (1ZY7) [ 50 ] and CD14 (1WWL) [ 35 ]; red (acidic) and blue (basic) are according to the electrostatic potential. IP 6 potentially interacts with CD14 residues R92, R97, R150, and R230. All comparisons were performed by one-way ANOVA with Tukey’s post-hoc multiple comparison test (Fig. 1C) or unpaired student t test (Fig. 1A). All data are presented as mean ± standard deviation (SD) for each group. * p
    Figure Legend Snippet: The role of CD14 + macrophages in IC-IP 6 uptake and PIP recognition. Representative liver sections (left) and quantitative data (right) from mice treated with or without intravenous IC-IP 6 ( n = 6 per group). The liver sections were stained with Prussian blue (PB) for iron and anti-F4/80 antibodies for macrophages/Kupffer cells (scale bars, 20 μm). F4/80+ PB+ cells were quantified using ImageJ software. B , C LPS induction of IC-IP 6 uptake by TPMs, determined by PB-stained cells (scale bars, 20 μm) ( B ), or ( C ) by iron staining with 2,4,6-tri-(2-pyridyl)-5-triazine (TPTZ) in cell lysates ( n = 8). Each point represents the mean of three experimental replicates for each IC-IP 6 concentration. D Proteins isolated from RAW264.7 cell lysates by IC-IP 6 precipitation were eluted with NaCl, separated by SDS-PAGE, and visualized using Coomassie Brilliant Blue (black arrowhead = CD14). CD14 identity was confirmed by western blotting. E , F CD14 binding to immobilized phospholipids (100 pmol each) ( E ), and varying concentrations of phosphatidylinositol phosphates (PIP strips or PIP arrays, Echelon Biosciences) ( F ). G Representative flow cytometry analyses of the binding of CD14, AKT PHD, or anti-PIP 3 antibody to the indicated phospholipids. Silica particles loaded with specific combinations of phospholipids (Echelon Bioscience) were treated with anti-PI(3,4,5)P 3 antibody or His-tagged AKT PHD, or CD14 protein. Samples with His-tagged proteins were treated with primary anti-His antibody, and all were visualized using an Alexa-647-labeled secondary antibody. The blue dotted lines indicate the peaks of the isotype controls. H ITC results for IP 6 titration into 30 μM CD14; the K d value was determined by curve fitting the raw data ( n = 2; MicroCal). I Modeled PI(3,4,5)P 3 -CD14 structure, generated with AutoDock PyRx and coordinates for IP 6 (1ZY7) [ 50 ] and CD14 (1WWL) [ 35 ]; red (acidic) and blue (basic) are according to the electrostatic potential. IP 6 potentially interacts with CD14 residues R92, R97, R150, and R230. All comparisons were performed by one-way ANOVA with Tukey’s post-hoc multiple comparison test (Fig. 1C) or unpaired student t test (Fig. 1A). All data are presented as mean ± standard deviation (SD) for each group. * p

    Techniques Used: Mouse Assay, Staining, Software, Concentration Assay, Isolation, SDS Page, Western Blot, Binding Assay, Flow Cytometry, Labeling, Titration, Generated, Standard Deviation

    In vivo induction of exofacial PIPs. A – D Flow cytometric analyses of thymocytes from mice 0–16 h after irradiation (10 Gy). A Representative flow cytometric plots (left) of propidium iodide (PI) and cells stained for caspase 3/7 covalent suicide inhibitor (Casp) showing the R1 (PI − Casp + ), R2 (PI + Casp + ), R3 (PI + Casp − ), and R4 (PI − Casp − ) subsets. Histograms (right) show the binding levels of Annexin V, AKT PHD, or CD14 in R1 (dark grey), R2 (red), R3 (light blue), and R4 (light grey) subsets ( n = 3). B Percentages of Annexin V, AKT PHD and CD14 binding for R1 (upper row), R2 (middle row) and R3 (lower row) cells. C Frequencies of Annexin V, AKT PHD, and CD14 in R1, R2, and R3 thymocytes. D The relative MFI values in R1, R2, and R3 cells for AKT PHD, Annexin V, and CD14 binding. The MFI values were normalized to those of R1 at each time point. E Representative images of jejunum sections from irradiated WT and Cd14 −/− mice were stained for cleaved caspase 3 (red), PI(3,4,5)P 3 (green), and DAPI (blue). F Representative images of pancreatic sections from mice treated with caerulein [ 47 ] stained with DAPI (blue), cleaved caspase 3 (red), and PI(3,4,5)P 3 (green). Scale bars, 10 μm. See also Supplementary Fig. 9 . All comparisons were performed by one-way ANOVA with Tukey’s post-hoc multiple comparison test. All data are presented as mean ± SD for each group ( n = 3 per group). * p
    Figure Legend Snippet: In vivo induction of exofacial PIPs. A – D Flow cytometric analyses of thymocytes from mice 0–16 h after irradiation (10 Gy). A Representative flow cytometric plots (left) of propidium iodide (PI) and cells stained for caspase 3/7 covalent suicide inhibitor (Casp) showing the R1 (PI − Casp + ), R2 (PI + Casp + ), R3 (PI + Casp − ), and R4 (PI − Casp − ) subsets. Histograms (right) show the binding levels of Annexin V, AKT PHD, or CD14 in R1 (dark grey), R2 (red), R3 (light blue), and R4 (light grey) subsets ( n = 3). B Percentages of Annexin V, AKT PHD and CD14 binding for R1 (upper row), R2 (middle row) and R3 (lower row) cells. C Frequencies of Annexin V, AKT PHD, and CD14 in R1, R2, and R3 thymocytes. D The relative MFI values in R1, R2, and R3 cells for AKT PHD, Annexin V, and CD14 binding. The MFI values were normalized to those of R1 at each time point. E Representative images of jejunum sections from irradiated WT and Cd14 −/− mice were stained for cleaved caspase 3 (red), PI(3,4,5)P 3 (green), and DAPI (blue). F Representative images of pancreatic sections from mice treated with caerulein [ 47 ] stained with DAPI (blue), cleaved caspase 3 (red), and PI(3,4,5)P 3 (green). Scale bars, 10 μm. See also Supplementary Fig. 9 . All comparisons were performed by one-way ANOVA with Tukey’s post-hoc multiple comparison test. All data are presented as mean ± SD for each group ( n = 3 per group). * p

    Techniques Used: In Vivo, Mouse Assay, Irradiation, Staining, Binding Assay

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    Echelon Biosciences anti phosphatidylinositol 3 4 5 trisphosphate pip3
    PDPK1 is activated in LRCC and silencing augments response to gemcitabine.  a  Immunoblots of LRCC and NLRCC fractions from MiaPaCa2, Panc-1, and Nor-P1 cells probed with anti-phospho PDPK1, anti-phospho AKT. Equal amounts of protein loaded, anti-AKT and β-Actin control on bottom.  b  Immunofluoresence of LRCC and NLRCC populations from MiaPaCa2, Panc-1, and Nor-P1 cells measuring anti-phosphatidylinositol 3,4,5-trisphosphate levels. Normalized mean of staining intensity for each population shown on the right.  c  Viability of pancreas cancer cells treated with gemcitabine alone (blue) or in combination with the PDPK1 inhibitor BX795 (purple) or AR-12 (red).  d  Induction of apoptosis in pancreas cancer cells treated with gemcitabine alone or in combination with the PDPK1 inhibitors BX795 and AR-12
    Anti Phosphatidylinositol 3 4 5 Trisphosphate Pip3, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti phosphatidylinositol 3 4 5 trisphosphate pip3/product/Echelon Biosciences
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    PDPK1 is activated in LRCC and silencing augments response to gemcitabine.  a  Immunoblots of LRCC and NLRCC fractions from MiaPaCa2, Panc-1, and Nor-P1 cells probed with anti-phospho PDPK1, anti-phospho AKT. Equal amounts of protein loaded, anti-AKT and β-Actin control on bottom.  b  Immunofluoresence of LRCC and NLRCC populations from MiaPaCa2, Panc-1, and Nor-P1 cells measuring anti-phosphatidylinositol 3,4,5-trisphosphate levels. Normalized mean of staining intensity for each population shown on the right.  c  Viability of pancreas cancer cells treated with gemcitabine alone (blue) or in combination with the PDPK1 inhibitor BX795 (purple) or AR-12 (red).  d  Induction of apoptosis in pancreas cancer cells treated with gemcitabine alone or in combination with the PDPK1 inhibitors BX795 and AR-12

    Journal: BMC Cancer

    Article Title: Loss of PDPK1 abrogates resistance to gemcitabine in label-retaining pancreatic cancer cells

    doi: 10.1186/s12885-018-4690-1

    Figure Lengend Snippet: PDPK1 is activated in LRCC and silencing augments response to gemcitabine. a Immunoblots of LRCC and NLRCC fractions from MiaPaCa2, Panc-1, and Nor-P1 cells probed with anti-phospho PDPK1, anti-phospho AKT. Equal amounts of protein loaded, anti-AKT and β-Actin control on bottom. b Immunofluoresence of LRCC and NLRCC populations from MiaPaCa2, Panc-1, and Nor-P1 cells measuring anti-phosphatidylinositol 3,4,5-trisphosphate levels. Normalized mean of staining intensity for each population shown on the right. c Viability of pancreas cancer cells treated with gemcitabine alone (blue) or in combination with the PDPK1 inhibitor BX795 (purple) or AR-12 (red). d Induction of apoptosis in pancreas cancer cells treated with gemcitabine alone or in combination with the PDPK1 inhibitors BX795 and AR-12

    Article Snippet: Cells were permeabilized in 0.25% TritonX-100 and blocked with 5% normal goat serum in PBS at room temperature in a humidified chamber for 2 h. Slides were incubated anti-phosphatidylinositol 3,4,5-trisphosphate (PIP3) (Cat#Z-P345, Echelon Biosciences Inc., Salt Lake City, UT) monoclonal antibodies.

    Techniques: Western Blot, Staining

    Structural model for the differential effect of T280 phosphorylation on PIP3 vs. PI4P binding to the Grp1 PH domain. The phosphoinositide-binding site in the crystal structure of the Grp1 PH domain bound to the PIP3 head group (PDB ID 1FGY) is compared with a hypothetical composite model for the PI4P head group. The PI4P head group was acquired from the crystal structure of the ARNO PH domain bound to the PIP2 head group (PDB ID 1U29) after alignment of the PH domains and deletion of the 5-phosphate. Magenta dashes represent hydrogen bonds between the T280 side chain hydroxyl group and the 1-phosphate of the PIP3 head group that are not observed for the PIP2 head group in the ARNO complex.

    Journal: Molecular Biology of the Cell

    Article Title: Coordination of Grp1 recruitment mechanisms by its phosphorylation

    doi: 10.1091/mbc.E20-03-0173

    Figure Lengend Snippet: Structural model for the differential effect of T280 phosphorylation on PIP3 vs. PI4P binding to the Grp1 PH domain. The phosphoinositide-binding site in the crystal structure of the Grp1 PH domain bound to the PIP3 head group (PDB ID 1FGY) is compared with a hypothetical composite model for the PI4P head group. The PI4P head group was acquired from the crystal structure of the ARNO PH domain bound to the PIP2 head group (PDB ID 1U29) after alignment of the PH domains and deletion of the 5-phosphate. Magenta dashes represent hydrogen bonds between the T280 side chain hydroxyl group and the 1-phosphate of the PIP3 head group that are not observed for the PIP2 head group in the ARNO complex.

    Article Snippet: Newly acquired antibodies include mouse antibodies against PI4P (Z-P004) and PIP3 (Z-P345) from Echelon Biosciences (Salt Lake City, UT), mouse antibody against Rab11 (610656) from BD Transduction Laboratories (San Jose, CA), goat antibodies against GRASP (SC-55951) and IPCEF1 (SC-168195) from Santa Cruz (Dallas, TX), and anti-goat secondary antibodies conjugated to HRP (705-035-147) or Cy3 (705-165-147) from Jackson ImmunoResearch (West Grove, PA).

    Techniques: Binding Assay

    In vitro induction of exofacial PIPs. A , B Flow cytometric analyses of Jurkat cells treated 0–16 h with 10 μM camptothecin. A Representative flow cytometric plots (left) of TO-PRO-3 (TP-3) and propidium iodide (PI)-stained cells showing R1 (TP-3 + PI − ), R2 (TP-3 + PI + ), R3 (TP-3 − PI + ), and R4 (TP-3 − PI − ) subsets. Histograms (right) show the binding levels of Annexin V, AKT PHD, CD14, or PIP 3 antibody in R1 (dark gray), R2 (red), and R4 (light grey) subsets. B Percentage of Annexin V, AKT PHD, CD14, and PI(3,4,5)P 3 in R1 (upper low) and R2 (lower low) cells. C , D Flow cytometric analyses of Jurkat cells treated 16 h with 10 μM camptothecin. C Representative flow cytometric plots (left) and binding percentages (right) of an AKT PHD and CD14 combination. D Representative flow cytometric plots (left) and binding percentages (right) of an Annexin V and AKT PHD combination. E Representative flow cytometric plots (left) and binding percentages (right) of an Annexin V and CD14 combination. F CHO cells treated 6 h with 10 μM camptothecin and incubated with Annexin V and AKT PHD were detected with primary antibodies and Texas Red- or FITC-labeled secondary antibodies, respectively. See also Supplementary Fig. 8 and Supplementary Movie 2 . All comparisons were performed by one-way ANOVA with Tukey’s post-hoc multiple comparison test. All data are presented as the mean ± SD for each group ( n = 6 per group unless noted otherwise). * p

    Journal: Cell Death and Differentiation

    Article Title: Externalized phosphatidylinositides on apoptotic cells are eat-me signals recognized by CD14

    doi: 10.1038/s41418-022-00931-2

    Figure Lengend Snippet: In vitro induction of exofacial PIPs. A , B Flow cytometric analyses of Jurkat cells treated 0–16 h with 10 μM camptothecin. A Representative flow cytometric plots (left) of TO-PRO-3 (TP-3) and propidium iodide (PI)-stained cells showing R1 (TP-3 + PI − ), R2 (TP-3 + PI + ), R3 (TP-3 − PI + ), and R4 (TP-3 − PI − ) subsets. Histograms (right) show the binding levels of Annexin V, AKT PHD, CD14, or PIP 3 antibody in R1 (dark gray), R2 (red), and R4 (light grey) subsets. B Percentage of Annexin V, AKT PHD, CD14, and PI(3,4,5)P 3 in R1 (upper low) and R2 (lower low) cells. C , D Flow cytometric analyses of Jurkat cells treated 16 h with 10 μM camptothecin. C Representative flow cytometric plots (left) and binding percentages (right) of an AKT PHD and CD14 combination. D Representative flow cytometric plots (left) and binding percentages (right) of an Annexin V and AKT PHD combination. E Representative flow cytometric plots (left) and binding percentages (right) of an Annexin V and CD14 combination. F CHO cells treated 6 h with 10 μM camptothecin and incubated with Annexin V and AKT PHD were detected with primary antibodies and Texas Red- or FITC-labeled secondary antibodies, respectively. See also Supplementary Fig. 8 and Supplementary Movie 2 . All comparisons were performed by one-way ANOVA with Tukey’s post-hoc multiple comparison test. All data are presented as the mean ± SD for each group ( n = 6 per group unless noted otherwise). * p

    Article Snippet: After fixation, the cells were treated with serum-free protein blocking solution (DAKO) and incubated with anti-PIP3 IgM antibody (Z-P345, 1:100, Echelon Biosciences), CD14, AKT PHD, AKT-PHD/eGFP, or Annexin V. For immunofluorescence studies, tissue sections (2.5 μm) were blocked (DAKO) and incubated with anti-PI(3,4,5)P3 and anti-cleaved caspase 3 antibodies.

    Techniques: In Vitro, Staining, Binding Assay, Incubation, Labeling

    PIP externalization and its role in phagocytosis. A Representative images of apoptotic cell detection by recombinant WT CD14 or mutant R4A CD14 protein. After inducing apoptosis with an anti-FAS antibody (100 ng/ml), HeLa cells incubated with recombinant WT or R4A CD14 were visualized using an anti-CD14 primary antibody and an FITC-labeled secondary antibody (scale bars, 10 μm). B Representative images of TPM phagocytosis of camptothecin-treated, pHrodo-labeled Jurkat cells using an antibody or recombinant proteins to mask externalized PS or PIPs. TPM phagocytosis of camptothecin-treated, pHrodo-labeled Jurkat cells was inhibited by an anti-PI(3,4,5)P 3 antibody, AKT PHD, Annexin V, and CD14 (scale bars, 50 μm). The red color represents engulfed apoptotic cells labeled with pHrodo dyes. C TPM phagocytosis of camptothecin-treated, pHrodo-labeled Jurkat cells was quantified in 8–10 low-power fields (mean ± SD, n = 5–10). D Representative flow cytometric plots (left) and percentages (right) of F4/80 + TPM phagocytosis of pHrodo-labeled thymocytes isolated from irradiated WT or Cd14 −/− mice group ( n = 6 per group). Thymocytes from irradiated mice were labeled with pHrodo for 30 min prior to adding TPMs from WT or Cd14 −/− mice for 1 hr (mean ± SD, n = 6). E Representative flow cytometry plots (upper) and MFI values (lower) of in vivo phagocytosis of pHrodo-labeled apoptotic thymocytes by F4/80+ mouse peritoneal macrophages of WT or Cd14 −/− mice group ( n = 3 per group). Thymocytes from irradiated mice were labeled with pHrodo for 30 min prior to injecting the peritoneal cavity of WT or Cd14 −/− mice for 15 min. F Representative fluorescent images (left) and intensities (right) of engulfed synthetic Bodipy-labeled PI(3,4,5)P 3 in TPMs from WT or Cd14 −/− mice. Engulfed Bodipy-labeled PIP 3 was visualized by confocal microscopy and quantified by fluorescence intensity using NIH ImageJ software (mean ± SD, n = 10) after incubating the TPMs with 1 μg of Bodipy-labeled PI(3,4,5)P 3 per well for 30 min (scale bars, 10 μm). G Representative flow cytometric plots (left) and MFI values (right) of engulfed synthetic Bodipy-labeled PI(3,4,5)P 3 in PI - /F4/80 + TPMs from WT or Cd14 −/− mice (mean ± SD, n = 6). All comparisons were performed by one-way ANOVA with Tukey’s post-hoc multiple comparison test Fig. 3A) or unpaired student t tests (Fig. 4D–F ). All data are presented as the mean ± SD for each group. * p

    Journal: Cell Death and Differentiation

    Article Title: Externalized phosphatidylinositides on apoptotic cells are eat-me signals recognized by CD14

    doi: 10.1038/s41418-022-00931-2

    Figure Lengend Snippet: PIP externalization and its role in phagocytosis. A Representative images of apoptotic cell detection by recombinant WT CD14 or mutant R4A CD14 protein. After inducing apoptosis with an anti-FAS antibody (100 ng/ml), HeLa cells incubated with recombinant WT or R4A CD14 were visualized using an anti-CD14 primary antibody and an FITC-labeled secondary antibody (scale bars, 10 μm). B Representative images of TPM phagocytosis of camptothecin-treated, pHrodo-labeled Jurkat cells using an antibody or recombinant proteins to mask externalized PS or PIPs. TPM phagocytosis of camptothecin-treated, pHrodo-labeled Jurkat cells was inhibited by an anti-PI(3,4,5)P 3 antibody, AKT PHD, Annexin V, and CD14 (scale bars, 50 μm). The red color represents engulfed apoptotic cells labeled with pHrodo dyes. C TPM phagocytosis of camptothecin-treated, pHrodo-labeled Jurkat cells was quantified in 8–10 low-power fields (mean ± SD, n = 5–10). D Representative flow cytometric plots (left) and percentages (right) of F4/80 + TPM phagocytosis of pHrodo-labeled thymocytes isolated from irradiated WT or Cd14 −/− mice group ( n = 6 per group). Thymocytes from irradiated mice were labeled with pHrodo for 30 min prior to adding TPMs from WT or Cd14 −/− mice for 1 hr (mean ± SD, n = 6). E Representative flow cytometry plots (upper) and MFI values (lower) of in vivo phagocytosis of pHrodo-labeled apoptotic thymocytes by F4/80+ mouse peritoneal macrophages of WT or Cd14 −/− mice group ( n = 3 per group). Thymocytes from irradiated mice were labeled with pHrodo for 30 min prior to injecting the peritoneal cavity of WT or Cd14 −/− mice for 15 min. F Representative fluorescent images (left) and intensities (right) of engulfed synthetic Bodipy-labeled PI(3,4,5)P 3 in TPMs from WT or Cd14 −/− mice. Engulfed Bodipy-labeled PIP 3 was visualized by confocal microscopy and quantified by fluorescence intensity using NIH ImageJ software (mean ± SD, n = 10) after incubating the TPMs with 1 μg of Bodipy-labeled PI(3,4,5)P 3 per well for 30 min (scale bars, 10 μm). G Representative flow cytometric plots (left) and MFI values (right) of engulfed synthetic Bodipy-labeled PI(3,4,5)P 3 in PI - /F4/80 + TPMs from WT or Cd14 −/− mice (mean ± SD, n = 6). All comparisons were performed by one-way ANOVA with Tukey’s post-hoc multiple comparison test Fig. 3A) or unpaired student t tests (Fig. 4D–F ). All data are presented as the mean ± SD for each group. * p

    Article Snippet: After fixation, the cells were treated with serum-free protein blocking solution (DAKO) and incubated with anti-PIP3 IgM antibody (Z-P345, 1:100, Echelon Biosciences), CD14, AKT PHD, AKT-PHD/eGFP, or Annexin V. For immunofluorescence studies, tissue sections (2.5 μm) were blocked (DAKO) and incubated with anti-PI(3,4,5)P3 and anti-cleaved caspase 3 antibodies.

    Techniques: Recombinant, Mutagenesis, Incubation, Labeling, Isolation, Irradiation, Mouse Assay, Flow Cytometry, In Vivo, Confocal Microscopy, Fluorescence, Software

    The role of CD14 + macrophages in IC-IP 6 uptake and PIP recognition. Representative liver sections (left) and quantitative data (right) from mice treated with or without intravenous IC-IP 6 ( n = 6 per group). The liver sections were stained with Prussian blue (PB) for iron and anti-F4/80 antibodies for macrophages/Kupffer cells (scale bars, 20 μm). F4/80+ PB+ cells were quantified using ImageJ software. B , C LPS induction of IC-IP 6 uptake by TPMs, determined by PB-stained cells (scale bars, 20 μm) ( B ), or ( C ) by iron staining with 2,4,6-tri-(2-pyridyl)-5-triazine (TPTZ) in cell lysates ( n = 8). Each point represents the mean of three experimental replicates for each IC-IP 6 concentration. D Proteins isolated from RAW264.7 cell lysates by IC-IP 6 precipitation were eluted with NaCl, separated by SDS-PAGE, and visualized using Coomassie Brilliant Blue (black arrowhead = CD14). CD14 identity was confirmed by western blotting. E , F CD14 binding to immobilized phospholipids (100 pmol each) ( E ), and varying concentrations of phosphatidylinositol phosphates (PIP strips or PIP arrays, Echelon Biosciences) ( F ). G Representative flow cytometry analyses of the binding of CD14, AKT PHD, or anti-PIP 3 antibody to the indicated phospholipids. Silica particles loaded with specific combinations of phospholipids (Echelon Bioscience) were treated with anti-PI(3,4,5)P 3 antibody or His-tagged AKT PHD, or CD14 protein. Samples with His-tagged proteins were treated with primary anti-His antibody, and all were visualized using an Alexa-647-labeled secondary antibody. The blue dotted lines indicate the peaks of the isotype controls. H ITC results for IP 6 titration into 30 μM CD14; the K d value was determined by curve fitting the raw data ( n = 2; MicroCal). I Modeled PI(3,4,5)P 3 -CD14 structure, generated with AutoDock PyRx and coordinates for IP 6 (1ZY7) [ 50 ] and CD14 (1WWL) [ 35 ]; red (acidic) and blue (basic) are according to the electrostatic potential. IP 6 potentially interacts with CD14 residues R92, R97, R150, and R230. All comparisons were performed by one-way ANOVA with Tukey’s post-hoc multiple comparison test (Fig. 1C) or unpaired student t test (Fig. 1A). All data are presented as mean ± standard deviation (SD) for each group. * p

    Journal: Cell Death and Differentiation

    Article Title: Externalized phosphatidylinositides on apoptotic cells are eat-me signals recognized by CD14

    doi: 10.1038/s41418-022-00931-2

    Figure Lengend Snippet: The role of CD14 + macrophages in IC-IP 6 uptake and PIP recognition. Representative liver sections (left) and quantitative data (right) from mice treated with or without intravenous IC-IP 6 ( n = 6 per group). The liver sections were stained with Prussian blue (PB) for iron and anti-F4/80 antibodies for macrophages/Kupffer cells (scale bars, 20 μm). F4/80+ PB+ cells were quantified using ImageJ software. B , C LPS induction of IC-IP 6 uptake by TPMs, determined by PB-stained cells (scale bars, 20 μm) ( B ), or ( C ) by iron staining with 2,4,6-tri-(2-pyridyl)-5-triazine (TPTZ) in cell lysates ( n = 8). Each point represents the mean of three experimental replicates for each IC-IP 6 concentration. D Proteins isolated from RAW264.7 cell lysates by IC-IP 6 precipitation were eluted with NaCl, separated by SDS-PAGE, and visualized using Coomassie Brilliant Blue (black arrowhead = CD14). CD14 identity was confirmed by western blotting. E , F CD14 binding to immobilized phospholipids (100 pmol each) ( E ), and varying concentrations of phosphatidylinositol phosphates (PIP strips or PIP arrays, Echelon Biosciences) ( F ). G Representative flow cytometry analyses of the binding of CD14, AKT PHD, or anti-PIP 3 antibody to the indicated phospholipids. Silica particles loaded with specific combinations of phospholipids (Echelon Bioscience) were treated with anti-PI(3,4,5)P 3 antibody or His-tagged AKT PHD, or CD14 protein. Samples with His-tagged proteins were treated with primary anti-His antibody, and all were visualized using an Alexa-647-labeled secondary antibody. The blue dotted lines indicate the peaks of the isotype controls. H ITC results for IP 6 titration into 30 μM CD14; the K d value was determined by curve fitting the raw data ( n = 2; MicroCal). I Modeled PI(3,4,5)P 3 -CD14 structure, generated with AutoDock PyRx and coordinates for IP 6 (1ZY7) [ 50 ] and CD14 (1WWL) [ 35 ]; red (acidic) and blue (basic) are according to the electrostatic potential. IP 6 potentially interacts with CD14 residues R92, R97, R150, and R230. All comparisons were performed by one-way ANOVA with Tukey’s post-hoc multiple comparison test (Fig. 1C) or unpaired student t test (Fig. 1A). All data are presented as mean ± standard deviation (SD) for each group. * p

    Article Snippet: After fixation, the cells were treated with serum-free protein blocking solution (DAKO) and incubated with anti-PIP3 IgM antibody (Z-P345, 1:100, Echelon Biosciences), CD14, AKT PHD, AKT-PHD/eGFP, or Annexin V. For immunofluorescence studies, tissue sections (2.5 μm) were blocked (DAKO) and incubated with anti-PI(3,4,5)P3 and anti-cleaved caspase 3 antibodies.

    Techniques: Mouse Assay, Staining, Software, Concentration Assay, Isolation, SDS Page, Western Blot, Binding Assay, Flow Cytometry, Labeling, Titration, Generated, Standard Deviation

    In vivo induction of exofacial PIPs. A – D Flow cytometric analyses of thymocytes from mice 0–16 h after irradiation (10 Gy). A Representative flow cytometric plots (left) of propidium iodide (PI) and cells stained for caspase 3/7 covalent suicide inhibitor (Casp) showing the R1 (PI − Casp + ), R2 (PI + Casp + ), R3 (PI + Casp − ), and R4 (PI − Casp − ) subsets. Histograms (right) show the binding levels of Annexin V, AKT PHD, or CD14 in R1 (dark grey), R2 (red), R3 (light blue), and R4 (light grey) subsets ( n = 3). B Percentages of Annexin V, AKT PHD and CD14 binding for R1 (upper row), R2 (middle row) and R3 (lower row) cells. C Frequencies of Annexin V, AKT PHD, and CD14 in R1, R2, and R3 thymocytes. D The relative MFI values in R1, R2, and R3 cells for AKT PHD, Annexin V, and CD14 binding. The MFI values were normalized to those of R1 at each time point. E Representative images of jejunum sections from irradiated WT and Cd14 −/− mice were stained for cleaved caspase 3 (red), PI(3,4,5)P 3 (green), and DAPI (blue). F Representative images of pancreatic sections from mice treated with caerulein [ 47 ] stained with DAPI (blue), cleaved caspase 3 (red), and PI(3,4,5)P 3 (green). Scale bars, 10 μm. See also Supplementary Fig. 9 . All comparisons were performed by one-way ANOVA with Tukey’s post-hoc multiple comparison test. All data are presented as mean ± SD for each group ( n = 3 per group). * p

    Journal: Cell Death and Differentiation

    Article Title: Externalized phosphatidylinositides on apoptotic cells are eat-me signals recognized by CD14

    doi: 10.1038/s41418-022-00931-2

    Figure Lengend Snippet: In vivo induction of exofacial PIPs. A – D Flow cytometric analyses of thymocytes from mice 0–16 h after irradiation (10 Gy). A Representative flow cytometric plots (left) of propidium iodide (PI) and cells stained for caspase 3/7 covalent suicide inhibitor (Casp) showing the R1 (PI − Casp + ), R2 (PI + Casp + ), R3 (PI + Casp − ), and R4 (PI − Casp − ) subsets. Histograms (right) show the binding levels of Annexin V, AKT PHD, or CD14 in R1 (dark grey), R2 (red), R3 (light blue), and R4 (light grey) subsets ( n = 3). B Percentages of Annexin V, AKT PHD and CD14 binding for R1 (upper row), R2 (middle row) and R3 (lower row) cells. C Frequencies of Annexin V, AKT PHD, and CD14 in R1, R2, and R3 thymocytes. D The relative MFI values in R1, R2, and R3 cells for AKT PHD, Annexin V, and CD14 binding. The MFI values were normalized to those of R1 at each time point. E Representative images of jejunum sections from irradiated WT and Cd14 −/− mice were stained for cleaved caspase 3 (red), PI(3,4,5)P 3 (green), and DAPI (blue). F Representative images of pancreatic sections from mice treated with caerulein [ 47 ] stained with DAPI (blue), cleaved caspase 3 (red), and PI(3,4,5)P 3 (green). Scale bars, 10 μm. See also Supplementary Fig. 9 . All comparisons were performed by one-way ANOVA with Tukey’s post-hoc multiple comparison test. All data are presented as mean ± SD for each group ( n = 3 per group). * p

    Article Snippet: After fixation, the cells were treated with serum-free protein blocking solution (DAKO) and incubated with anti-PIP3 IgM antibody (Z-P345, 1:100, Echelon Biosciences), CD14, AKT PHD, AKT-PHD/eGFP, or Annexin V. For immunofluorescence studies, tissue sections (2.5 μm) were blocked (DAKO) and incubated with anti-PI(3,4,5)P3 and anti-cleaved caspase 3 antibodies.

    Techniques: In Vivo, Mouse Assay, Irradiation, Staining, Binding Assay