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fluorescein conjugated anti pi (Echelon Biosciences)

Name: fluorescein conjugated anti pi
Brand: Echelon Biosciences
Rating: 93 (38 reviews)

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Echelon Biosciences fluorescein conjugated anti pi
Fluorescein Conjugated Anti Pi, 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
https://www.bioz.com/result/fluorescein conjugated anti pi/product/Echelon Biosciences
Average 93 stars, based on 1 article reviews

fluorescein conjugated anti pi - by Bioz Stars, 2025-03

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A–C A mass ELISA was used to detect specific PIP species after exposure to anti‐FcγRII/III (2.4G2) with or without 3‐a‐aminocholestane (SHIP 1 inhibitor), SF1670 (PTEN inhibitor) and LY294002 (PI‐3K inhibitor). In M‐MOP cells (left panel) and primary microglia (right panel), PI(4,5)P 2 (A), PI(3,4)P 2 (B) and PIP 3 (C) were detected. All data show the mean ± SD of three independent experiments and were analysed by two‐way ANOVA (log‐transformed data were used in C) with Sidak’s multiple comparison tests performed. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 (blue: P522 and red: R522).

Journal: The EMBO Journal

Article Title: PIP2 depletion and altered endocytosis caused by expression of Alzheimer's disease‐protective variant PLCγ2 R522

doi: 10.15252/embj.2020105603

Figure Lengend Snippet: A–C A mass ELISA was used to detect specific PIP species after exposure to anti‐FcγRII/III (2.4G2) with or without 3‐a‐aminocholestane (SHIP 1 inhibitor), SF1670 (PTEN inhibitor) and LY294002 (PI‐3K inhibitor). In M‐MOP cells (left panel) and primary microglia (right panel), PI(4,5)P 2 (A), PI(3,4)P 2 (B) and PIP 3 (C) were detected. All data show the mean ± SD of three independent experiments and were analysed by two‐way ANOVA (log‐transformed data were used in C) with Sidak’s multiple comparison tests performed. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 (blue: P522 and red: R522).

Article Snippet: Cells were then stained overnight at 4°C with anti‐PI(4,5)P2 IgM (Z‐G045 Echelon Biosciences 1:100) and anti‐IBA1 (013‐26471 Alphalabs 1:200).

Techniques: Enzyme-linked Immunosorbent Assay, Transformation Assay

$A Schematic of live‐cell dextran and transferrin pulse‐chase in degron‐ORP2 cells. B Exemplary confocal images from movies of +/− IAA‐treated degron‐ORP2 cells labeled with AF647‐transferrin (magenta) and fluorescein dextran (green) as in Fig . Trajectories of manually tracked double‐positive organelles at 30 min chase time point of the corresponding time series shown in Movie (‐IAA; ORP2 present) and Movie (+IAA; ORP2 depleted). C Duration of contacts between transferrin and dextran organelles. Mean ± SEM, n = 28 contacts from 6 cells (+IAA); 36 contacts from 5 cells (‐IAA). Student’s t ‐test. Number of contacts between dextran and transferrin organelles. Mean ± SEM. n = 42 (‐IAA); 39 (+IAA) dextran organelles. Student’s t ‐test. D Control and degron‐ORP2 cells were co‐plated. After 1 day in 5% LPDS, cells were loaded with 50 μg/ml LDL and IAA for the indicated times, stained with PI(4,5)P 2 antibody, and imaged by confocal microscopy. Asterisk indicates ORP2‐depleted cell. E Quantification of EV5D. Mean ± SD, n = 13–23 cells from 2 independent experiments. Student’s t ‐test. F, G TIRF imaging of NPC1‐GFP distribution in live cells with or without 10 μM PF‐228 for 4 h, and quantification of NPC1 vesicles within 10 μm distance from the cell edge (dotted lines). Transiently transfected mCherry‐FAK was used to mark the cell edge. Mean ± SD, n = 6 cells. Student’s t ‐test. H Representative epifluorescent images of NPC1‐mCherry organelles in GFP‐ORP2 or GFP‐ORP2‐mHHK overexpressing cells quantified in Fig . Arrowheads indicate tubular NPC1 organelles. I After 1 day in 5% LPDS, control and degron‐ORP2 cells were loaded with 50 μg/ml LDL and IAA for the indicated times, lysed and subjected to immunoblotting with pFAK antibody. Mean ± SD, n = 3 independent experiments. Paired Student’s t ‐test. J Control and degron‐ORP2 cells were co‐plated and incubated without (−LDL) or with LDL (+LDL) and IAA (+IAA; ORP2 depletion) for 1 h. Representative confocal images showing increased pFAK intensity at perinuclear endomembranes upon LDL loading in control cells but not in the ORP2‐depleted degron cell (asterisks). Red inset shows endomembrane endo‐GFP‐ORP2 and pFAK signals in the control cell, and blue inset shows corresponding signals in the ORP2‐depleted cell. Orange arrowheads indicate mature FAs. K Representative confocal images of GFP‐ORP2‐mHHK or GFP‐ORP2‐∆ELSK‐transfected cells quantified in Fig . Dashed lines indicate cell outlines. Asterisks indicate cells depleted of endogenous ORP2. L AF488‐FERM binding to liposomes by liposome‐co‐sedimentation. Lipid composition and concentration as well as protein concentration were the same as in Fig , in 0, 5, and 10% PI(4,5)P 2 ‐containing liposomes. S; supernatant, P; pellet. Numbers under the blots indicate the fraction of FAK FERM bound to liposomes (P) of total FAK FERM (S + P). Source data are available online for this figure.$

Journal: The EMBO Journal

Article Title: ORP2 couples LDL‐cholesterol transport to FAK activation by endosomal cholesterol/PI(4,5)P 2 exchange

doi: 10.15252/embj.2020106871

Figure Lengend Snippet: A Schematic of live‐cell dextran and transferrin pulse‐chase in degron‐ORP2 cells. B Exemplary confocal images from movies of +/− IAA‐treated degron‐ORP2 cells labeled with AF647‐transferrin (magenta) and fluorescein dextran (green) as in Fig . Trajectories of manually tracked double‐positive organelles at 30 min chase time point of the corresponding time series shown in Movie (‐IAA; ORP2 present) and Movie (+IAA; ORP2 depleted). C Duration of contacts between transferrin and dextran organelles. Mean ± SEM, n = 28 contacts from 6 cells (+IAA); 36 contacts from 5 cells (‐IAA). Student’s t ‐test. Number of contacts between dextran and transferrin organelles. Mean ± SEM. n = 42 (‐IAA); 39 (+IAA) dextran organelles. Student’s t ‐test. D Control and degron‐ORP2 cells were co‐plated. After 1 day in 5% LPDS, cells were loaded with 50 μg/ml LDL and IAA for the indicated times, stained with PI(4,5)P 2 antibody, and imaged by confocal microscopy. Asterisk indicates ORP2‐depleted cell. E Quantification of EV5D. Mean ± SD, n = 13–23 cells from 2 independent experiments. Student’s t ‐test. F, G TIRF imaging of NPC1‐GFP distribution in live cells with or without 10 μM PF‐228 for 4 h, and quantification of NPC1 vesicles within 10 μm distance from the cell edge (dotted lines). Transiently transfected mCherry‐FAK was used to mark the cell edge. Mean ± SD, n = 6 cells. Student’s t ‐test. H Representative epifluorescent images of NPC1‐mCherry organelles in GFP‐ORP2 or GFP‐ORP2‐mHHK overexpressing cells quantified in Fig . Arrowheads indicate tubular NPC1 organelles. I After 1 day in 5% LPDS, control and degron‐ORP2 cells were loaded with 50 μg/ml LDL and IAA for the indicated times, lysed and subjected to immunoblotting with pFAK antibody. Mean ± SD, n = 3 independent experiments. Paired Student’s t ‐test. J Control and degron‐ORP2 cells were co‐plated and incubated without (−LDL) or with LDL (+LDL) and IAA (+IAA; ORP2 depletion) for 1 h. Representative confocal images showing increased pFAK intensity at perinuclear endomembranes upon LDL loading in control cells but not in the ORP2‐depleted degron cell (asterisks). Red inset shows endomembrane endo‐GFP‐ORP2 and pFAK signals in the control cell, and blue inset shows corresponding signals in the ORP2‐depleted cell. Orange arrowheads indicate mature FAs. K Representative confocal images of GFP‐ORP2‐mHHK or GFP‐ORP2‐∆ELSK‐transfected cells quantified in Fig . Dashed lines indicate cell outlines. Asterisks indicate cells depleted of endogenous ORP2. L AF488‐FERM binding to liposomes by liposome‐co‐sedimentation. Lipid composition and concentration as well as protein concentration were the same as in Fig , in 0, 5, and 10% PI(4,5)P 2 ‐containing liposomes. S; supernatant, P; pellet. Numbers under the blots indicate the fraction of FAK FERM bound to liposomes (P) of total FAK FERM (S + P). Source data are available online for this figure.

Article Snippet: Antibodies: pFAK (BD Transduction Laboratory #611807, clone 18 for Western blotting and Thermo Fisher #44‐624G for immunofluorescence), FAK (Sigma 05‐537, Clone 4.47), integrin β1 (Santa Cruz sc‐18887, K20 for immunofluorescence and Sigma MAB2252, clone N29 for Western blotting), PI(4,5)P 2 (Echelon, Z‐G045, clone 2C11), ORP2 (Novus Biologicals, NBP1‐92236), NPC1 (Abcam, ab134113), Lamp1 (DSHB, H4A3), GFP (Abcam, ab290 and ab1218); see Appendix␣Fig for specificity controls of pFAK, FAK, and integrin β1antibodies.␣Lipids: 1‐palmitoyl‐2oleoyl‐sn‐ glycero‐3‐phosphocholine (POPC), 1‐palmitoyl‐2oleoyl‐sn‐glycero‐3‐phosphoethanolamine (POPE), 1‐palmitoyl‐2oleoyl‐sn‐glycero‐3‐phospho‐L‐serine (sodium salt) (POPS), L‐a‐phosphatidylinositol‐4,5‐bisphosphate (brain, porcine, ammonium salt; brain PI(4,5)P 2 ), cholesterol (ovine) were from Avanti Polar Lipids.

Techniques: Pulse Chase, Labeling, Staining, Confocal Microscopy, Imaging, Transfection, Western Blot, Incubation, Binding Assay, Sedimentation, Concentration Assay, Protein Concentration

A After 1 day in 5% LPDS, NPC1‐GFP expressing cells were loaded with 50 μg/ml LDL with or without 10 μM PF‐228 for the indicated times, and stained for intracellular D4H. Representative confocal images of intracellular D4H, NPC1‐GFP, and internalized integrin β1 labeling. Dashed lines indicate cell outlines. B Fraction of D4H‐positive area in the cell as in (A). C Fraction of D4H area overlapping with NPC1 in a peripheral region toward the leading edge (exemplary box shown as inset in (A)). D Quantification of D4H area positive for integrin β1 in a peripheral region toward the leading edge. Mean ± SD, n = 26–30 cells pooled from 2 independent experiments. Student’s t ‐test. E–G Control and FAK siRNA‐treated cells were LDL depleted for 1 day, loaded with LDL for the indicated times, and stained for plasma membrane D4H or for intracellular D4H with lamp1 or integrin β1 antibodies. Quantification of plasma membrane D4H (E) and intracellular D4H area positive for lamp1 (F) or integrin β1 (G); corresponding images in Fig . Mean ± SD, n = 20–35 cells from 2 independent experiments. Student’s t ‐test. H Schematic of FAK‐regulated PI(4,5)P 2 generation on endosomes via PIPKIγ, modified from (Nader et␣al , ). I After 1‐day 5% LPDS, cells were loaded with 50 μg/ml LDL with or without 10 μM PF‐228 for the indicated times and endomembrane PI(4,5)P 2 was stained with antibody and imaged by confocal microscopy. J Quantification of mean PI(4,5)P 2 immunoreactivity in cells. Mean ± SD, n = 16–26 cells pooled from 2 independent experiments. Student’s t ‐test.

Journal: The EMBO Journal

Article Title: ORP2 couples LDL‐cholesterol transport to FAK activation by endosomal cholesterol/PI(4,5)P 2 exchange

doi: 10.15252/embj.2020106871

Figure Lengend Snippet: A After 1 day in 5% LPDS, NPC1‐GFP expressing cells were loaded with 50 μg/ml LDL with or without 10 μM PF‐228 for the indicated times, and stained for intracellular D4H. Representative confocal images of intracellular D4H, NPC1‐GFP, and internalized integrin β1 labeling. Dashed lines indicate cell outlines. B Fraction of D4H‐positive area in the cell as in (A). C Fraction of D4H area overlapping with NPC1 in a peripheral region toward the leading edge (exemplary box shown as inset in (A)). D Quantification of D4H area positive for integrin β1 in a peripheral region toward the leading edge. Mean ± SD, n = 26–30 cells pooled from 2 independent experiments. Student’s t ‐test. E–G Control and FAK siRNA‐treated cells were LDL depleted for 1 day, loaded with LDL for the indicated times, and stained for plasma membrane D4H or for intracellular D4H with lamp1 or integrin β1 antibodies. Quantification of plasma membrane D4H (E) and intracellular D4H area positive for lamp1 (F) or integrin β1 (G); corresponding images in Fig . Mean ± SD, n = 20–35 cells from 2 independent experiments. Student’s t ‐test. H Schematic of FAK‐regulated PI(4,5)P 2 generation on endosomes via PIPKIγ, modified from (Nader et␣al , ). I After 1‐day 5% LPDS, cells were loaded with 50 μg/ml LDL with or without 10 μM PF‐228 for the indicated times and endomembrane PI(4,5)P 2 was stained with antibody and imaged by confocal microscopy. J Quantification of mean PI(4,5)P 2 immunoreactivity in cells. Mean ± SD, n = 16–26 cells pooled from 2 independent experiments. Student’s t ‐test.

Techniques: Expressing, Staining, Labeling, Modification, Confocal Microscopy

A Endo‐GFP‐ORP2 cells were treated with control or OCRL siRNAs for 2 days, stained with PI(4,5)P 2 antibody, and imaged by confocal microscopy. B Cells stably expressing NPC1‐mCherry were treated with control or OCRL siRNAs for 2 days, or transfected with GFP‐ORP2 or ‐ORP2‐mHHK (PI(4,5)P 2 binding‐deficient mutant) for 1 day, stained with PI(4,5)P 2 antibody, and imaged by confocal microscopy. For FAK inhibition, GFP‐ORP2 transfected cells were treated with 10 μM PF‐228 for 4 h. Dashed lines indicate cell edges. Arrowheads indicate colocalization of PI(4,5)P 2 and NPC1. C Quantification of (B). Values from control siRNA and Vector control were pooled and are shown as control siRNA/Vector control. Mean ± SD, n = 20–43 cells pooled from 2 independent experiments. Student’s t ‐test. D, E Cells stably expressing NPC1‐mCherry were transfected with GFP‐ORP2 or GFP‐ORP2‐mHHK for 1 day and were indicated, treated with 10 μM PF‐228 for 4 h. For degron‐ORP2 cells, NPC1‐mCherry was transiently transfected for 2 days and cells incubated without (‐IAA; ORP2 present) or with IAA (+IAA; ORP2 depleted) for 1 h. Arrowheads indicate tubular NPC1 organelles. Live cell images were acquired by widefield epifluorescence miroscopy and the longest NPC1 tubule per cell was measured. Mean ± SD, n = 102–122 cells. Students’s t ‐test. Please see also control (Movie ) and PF‐228‐treated cell (Movie ), both videos 1 min recordings with 1 s frame rate.

Journal: The EMBO Journal

Article Title: ORP2 couples LDL‐cholesterol transport to FAK activation by endosomal cholesterol/PI(4,5)P 2 exchange

doi: 10.15252/embj.2020106871

Figure Lengend Snippet: A Endo‐GFP‐ORP2 cells were treated with control or OCRL siRNAs for 2 days, stained with PI(4,5)P 2 antibody, and imaged by confocal microscopy. B Cells stably expressing NPC1‐mCherry were treated with control or OCRL siRNAs for 2 days, or transfected with GFP‐ORP2 or ‐ORP2‐mHHK (PI(4,5)P 2 binding‐deficient mutant) for 1 day, stained with PI(4,5)P 2 antibody, and imaged by confocal microscopy. For FAK inhibition, GFP‐ORP2 transfected cells were treated with 10 μM PF‐228 for 4 h. Dashed lines indicate cell edges. Arrowheads indicate colocalization of PI(4,5)P 2 and NPC1. C Quantification of (B). Values from control siRNA and Vector control were pooled and are shown as control siRNA/Vector control. Mean ± SD, n = 20–43 cells pooled from 2 independent experiments. Student’s t ‐test. D, E Cells stably expressing NPC1‐mCherry were transfected with GFP‐ORP2 or GFP‐ORP2‐mHHK for 1 day and were indicated, treated with 10 μM PF‐228 for 4 h. For degron‐ORP2 cells, NPC1‐mCherry was transiently transfected for 2 days and cells incubated without (‐IAA; ORP2 present) or with IAA (+IAA; ORP2 depleted) for 1 h. Arrowheads indicate tubular NPC1 organelles. Live cell images were acquired by widefield epifluorescence miroscopy and the longest NPC1 tubule per cell was measured. Mean ± SD, n = 102–122 cells. Students’s t ‐test. Please see also control (Movie ) and PF‐228‐treated cell (Movie ), both videos 1 min recordings with 1 s frame rate.

Techniques: Staining, Confocal Microscopy, Stable Transfection, Expressing, Transfection, Binding Assay, Mutagenesis, Inhibition, Plasmid Preparation, Incubation

$A, B Control and degron‐ORP2 cells were co‐plated, and after 1‐day 5% LPDS, treated with 50 μg/ml LDL and IAA (+IAA; ORP2 depletion) for the indicated times and immunostained for pFAK to quantify its intensity. For rescue experiments, degron‐ORP2 cells were plated and transfected with GFP‐ORP2 or ‐ORP2‐mHHK or ‐ORP2‐∆ELSK for 6 h. Cells expressing GFP‐ORP2 constructs at levels similar to endo‐GFP‐ORP2 were used for quantifying pFAK intensity. Dashed lines indicate cell outlines. See also Fig EV6C. Asterisk indicates cells depleted of endogenous ORP2. Images were acquired by confocal microscopy. Mean ± SD, n = 20–23 cells pooled from 2 independent experiments. Student’s t ‐test. C Quantification of FAK protein in cytosolic and membrane fractions in degron‐ORP2 cells with or without IAA. Cells were starved overnight in LPDS and loaded with 50 µg/mL LDL +/− IAA for 2 h. The proportion of FAK signal in the membrane fraction (of total FAK in cytosol + membranes) is presented ± SD. For no IAA n = 13 and for IAA n = 11, 2 independent experiments. Student’s t ‐test. D–F Liposome‐co‐sedimentation of FAK FERM with increasing concentrations of PI(4,5)P 2 and cholesterol. “S” in (D) indicates the supernatant and “P” pellet containing the FAK FERM‐bound to liposomes. The total lipid concentration was kept at 50 μM, and purified FAK FERM concentration at 1 μM. The initial lipid composition was POPC:POPE:POPS:Rhodamine‐DHPE:PI(4,5)P 2 :cholesterol (70:19:10:1:0:0, mol/mol). The increasing PI(4,5)P 2 and cholesterol concentrations were compensated by decreasing the amount of POPC. Mean ± SD, n = 5. Student’s t ‐test (* P < 0.05; ** P < 0.002). Numbers under the blots indicate fraction of FAK FERM bound to liposomes (pellet) of total FAK FERM (supernatant + pellet). G, H Binding of Alexa Fluor 488‐labeled FAK FERM domain to GUVs containing PI(4,5)P 2 and cholesterol. The lipid compositions were POPC:POPE:POPS:Rhodamine‐DHPE:PI(4,5)P 2 :cholesterol (50:19:10:1:20:0) and POPC:POPE:POPS:Rhodamine‐DHPE:PI(4,5)P 2 :cholesterol (20:19:10:1:20:30, mol/mol). Images were acquired by widefield epifluorescence microscopy. Scale bar, 5 μm. Mean ± SD, n = 68–76 pooled from 2 independent experiments. Student’s t ‐test. Source data are available online for this figure.$

Journal: The EMBO Journal

Article Title: ORP2 couples LDL‐cholesterol transport to FAK activation by endosomal cholesterol/PI(4,5)P 2 exchange

doi: 10.15252/embj.2020106871

Figure Lengend Snippet: A, B Control and degron‐ORP2 cells were co‐plated, and after 1‐day 5% LPDS, treated with 50 μg/ml LDL and IAA (+IAA; ORP2 depletion) for the indicated times and immunostained for pFAK to quantify its intensity. For rescue experiments, degron‐ORP2 cells were plated and transfected with GFP‐ORP2 or ‐ORP2‐mHHK or ‐ORP2‐∆ELSK for 6 h. Cells expressing GFP‐ORP2 constructs at levels similar to endo‐GFP‐ORP2 were used for quantifying pFAK intensity. Dashed lines indicate cell outlines. See also Fig EV6C. Asterisk indicates cells depleted of endogenous ORP2. Images were acquired by confocal microscopy. Mean ± SD, n = 20–23 cells pooled from 2 independent experiments. Student’s t ‐test. C Quantification of FAK protein in cytosolic and membrane fractions in degron‐ORP2 cells with or without IAA. Cells were starved overnight in LPDS and loaded with 50 µg/mL LDL +/− IAA for 2 h. The proportion of FAK signal in the membrane fraction (of total FAK in cytosol + membranes) is presented ± SD. For no IAA n = 13 and for IAA n = 11, 2 independent experiments. Student’s t ‐test. D–F Liposome‐co‐sedimentation of FAK FERM with increasing concentrations of PI(4,5)P 2 and cholesterol. “S” in (D) indicates the supernatant and “P” pellet containing the FAK FERM‐bound to liposomes. The total lipid concentration was kept at 50 μM, and purified FAK FERM concentration at 1 μM. The initial lipid composition was POPC:POPE:POPS:Rhodamine‐DHPE:PI(4,5)P 2 :cholesterol (70:19:10:1:0:0, mol/mol). The increasing PI(4,5)P 2 and cholesterol concentrations were compensated by decreasing the amount of POPC. Mean ± SD, n = 5. Student’s t ‐test (* P < 0.05; ** P < 0.002). Numbers under the blots indicate fraction of FAK FERM bound to liposomes (pellet) of total FAK FERM (supernatant + pellet). G, H Binding of Alexa Fluor 488‐labeled FAK FERM domain to GUVs containing PI(4,5)P 2 and cholesterol. The lipid compositions were POPC:POPE:POPS:Rhodamine‐DHPE:PI(4,5)P 2 :cholesterol (50:19:10:1:20:0) and POPC:POPE:POPS:Rhodamine‐DHPE:PI(4,5)P 2 :cholesterol (20:19:10:1:20:30, mol/mol). Images were acquired by widefield epifluorescence microscopy. Scale bar, 5 μm. Mean ± SD, n = 68–76 pooled from 2 independent experiments. Student’s t ‐test. Source data are available online for this figure.

Techniques: Transfection, Expressing, Construct, Confocal Microscopy, Sedimentation, Concentration Assay, Purification, Binding Assay, Labeling, Epifluorescence Microscopy

LDL‐cholesterol is taken up via LDL receptor‐mediated endocytosis and the liberated free cholesterol is incorporated into late endosomal limiting membrane via NPC2 and NPC1. ORP2 delivers cholesterol from late endosomes to FAK/integrin recycling endosomes where cholesterol facilitates FAK association with the PI(4,5)P 2 ‐containing membrane. Activated FAK increases the activity of endosomal PIPKIγ which generates PI(4,5)P 2 , accelerating the recycling of active integrins. ORP2 unloads PI(4,5)P 2 from FAK/integrin endosomes and delivers it to NPC1‐containing late endosomes, regulating their tubulovesicular dynamics. This coupling of ORP2 and FAK activity drives efficient cholesterol delivery to the plasma membrane, stimulating FA dynamics and cell migration.

Journal: The EMBO Journal

Article Title: ORP2 couples LDL‐cholesterol transport to FAK activation by endosomal cholesterol/PI(4,5)P 2 exchange

doi: 10.15252/embj.2020106871

Figure Lengend Snippet: LDL‐cholesterol is taken up via LDL receptor‐mediated endocytosis and the liberated free cholesterol is incorporated into late endosomal limiting membrane via NPC2 and NPC1. ORP2 delivers cholesterol from late endosomes to FAK/integrin recycling endosomes where cholesterol facilitates FAK association with the PI(4,5)P 2 ‐containing membrane. Activated FAK increases the activity of endosomal PIPKIγ which generates PI(4,5)P 2 , accelerating the recycling of active integrins. ORP2 unloads PI(4,5)P 2 from FAK/integrin endosomes and delivers it to NPC1‐containing late endosomes, regulating their tubulovesicular dynamics. This coupling of ORP2 and FAK activity drives efficient cholesterol delivery to the plasma membrane, stimulating FA dynamics and cell migration.

Techniques: Activity Assay, Migration

A mass ELISA was used to detect specific PIP species after exposure to anti-FcγRII/III (2.4G2) with or without 3-a-aminocholestane (SHIP 1 inhibitor), SF1670 (PTEN inhibitor) and LY294002 (PI-3K inhibitor). In M-MOP cells (left panel) and primary microglia (right panel) PI(4,5)P 2 (A) , PI(3,4)P 2 (B) and PIP 3 (C) were detected. All data shows the mean±SD of 3 independent experiments were analysed by 2 way ANOVA (log-transformed data was used in C) with Sidak’s multiple comparison tests performed *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. (blue: P522; red: R522)

Journal: bioRxiv

Article Title: The Alzheimer’s disease protective P522R variant of PLCG2 , consistently enhances stimulus-dependent PLCγ2 activation, depleting substrate and altering cell function

doi: 10.1101/2020.04.27.059600

Figure Lengend Snippet: A mass ELISA was used to detect specific PIP species after exposure to anti-FcγRII/III (2.4G2) with or without 3-a-aminocholestane (SHIP 1 inhibitor), SF1670 (PTEN inhibitor) and LY294002 (PI-3K inhibitor). In M-MOP cells (left panel) and primary microglia (right panel) PI(4,5)P 2 (A) , PI(3,4)P 2 (B) and PIP 3 (C) were detected. All data shows the mean±SD of 3 independent experiments were analysed by 2 way ANOVA (log-transformed data was used in C) with Sidak’s multiple comparison tests performed *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. (blue: P522; red: R522)

Article Snippet: Cells were then exposed to fluorescein conjugated Anti-PI(4,5)P2 IgM (Z-G045 Echelon Bioscience, 1:200, 4 hours).

Techniques: Enzyme-linked Immunosorbent Assay, Transformation Assay

Altered PI(4,5)P 2 subcellular distribution and receptor-mediated endocytosis in mPTCs derived from Dent disease mouse models. ( A ) Representative confocal micrographs and quantification of PI(4,5)P 2 + structures (green) in Ocrl mPTCs (n ≈ 80 cells pooled from 3 mouse kidneys per condition; each dot representing the number of PI ( , )P 2 + structures in a cell). ( B ) Representative confocal micrographs of Ocrl mPTCs immunostained with anti-PI(4,5)P 2 (green) and anti-EEA1 (red, early endosomes) and quantification (adjacent panel) of the number of PI(4,5)P 2 /EEA1 + structures by confocal microscopy (percentage of total EEA1 + vesicles: n = 3 Ocrl Y/+ and n = 4 Ocrl Y/− randomly selected fields per condition, each containing approximately 15–20 cells). Insets: high magnification of PI(4,5)P 2 /EEA1 + structures. ( C , D ) Ocrl and Clcn5 mPTCs were loaded with Alexa 488-BSA (green, 100 μg ml −1 for 15 min at 37°C), fixed and analyzed by confocal microscopy. Quantification of the number of Alexa 488-BSA + structures (n ≈ 150–250 cells pooled from 3 mouse kidneys per condition; each dot representing the number of BSA + structures in a cell). ( E ) Ocrl mPTCs were loaded with Alexa 647-dextran 10 kDa (red, 250 μg ml −1 for 30 min at 37°C), fixed and analyzed by confocal microscopy. Quantification of the number of Alexa 647-dextran + structures (n ≈ 200–250 cells pooled from 3 mouse kidneys per condition; each dot representing the number of dextran + structures in a cell). Nuclei counterstained with DAPI (blue). Scale bars: 15 μm in (A) and (B) and 10 μm in (C – E ) . Plotted data represent mean ± SEM. Two-tailed unpaired Student’s t -test, ** P < 0.01, *** P < 0.001 relative to Ocrl Y/+ or Clcn5 Y/+ mPTCs. ns: not significant.

Journal: Human Molecular Genetics

Article Title: OCRL deficiency impairs endolysosomal function in a humanized mouse model for Lowe syndrome and Dent disease

doi: 10.1093/hmg/ddy449

Figure Lengend Snippet: Altered PI(4,5)P 2 subcellular distribution and receptor-mediated endocytosis in mPTCs derived from Dent disease mouse models. ( A ) Representative confocal micrographs and quantification of PI(4,5)P 2 + structures (green) in Ocrl mPTCs (n ≈ 80 cells pooled from 3 mouse kidneys per condition; each dot representing the number of PI ( , )P 2 + structures in a cell). ( B ) Representative confocal micrographs of Ocrl mPTCs immunostained with anti-PI(4,5)P 2 (green) and anti-EEA1 (red, early endosomes) and quantification (adjacent panel) of the number of PI(4,5)P 2 /EEA1 + structures by confocal microscopy (percentage of total EEA1 + vesicles: n = 3 Ocrl Y/+ and n = 4 Ocrl Y/− randomly selected fields per condition, each containing approximately 15–20 cells). Insets: high magnification of PI(4,5)P 2 /EEA1 + structures. ( C , D ) Ocrl and Clcn5 mPTCs were loaded with Alexa 488-BSA (green, 100 μg ml −1 for 15 min at 37°C), fixed and analyzed by confocal microscopy. Quantification of the number of Alexa 488-BSA + structures (n ≈ 150–250 cells pooled from 3 mouse kidneys per condition; each dot representing the number of BSA + structures in a cell). ( E ) Ocrl mPTCs were loaded with Alexa 647-dextran 10 kDa (red, 250 μg ml −1 for 30 min at 37°C), fixed and analyzed by confocal microscopy. Quantification of the number of Alexa 647-dextran + structures (n ≈ 200–250 cells pooled from 3 mouse kidneys per condition; each dot representing the number of dextran + structures in a cell). Nuclei counterstained with DAPI (blue). Scale bars: 15 μm in (A) and (B) and 10 μm in (C – E ) . Plotted data represent mean ± SEM. Two-tailed unpaired Student’s t -test, ** P < 0.01, *** P < 0.001 relative to Ocrl Y/+ or Clcn5 Y/+ mPTCs. ns: not significant.

Article Snippet: The following antibodies were used: rabbit anti-human transferrin (A0061, Dako); rabbit anti-human Gc-globulin (also known as VDBP, A0021, Dako); rabbit anti-uteroglobin (also known as CC16, ab40873, Abcam); rabbit anti-SLC1A5 (also known as SGLT2, ab84903, Abcam); rabbit NaPi-IIa (gift from C.A.Wagner, University of Zurich, Zurich, Switzerland); rabbit anti-human AQP1 (ab2219, Millipore); mouse anti-β-actin (A2228, Sigma-Aldrich); mouse conjugated to Fluorescein (FITC) anti-PI(4,5)P 2 (Z-G045, Echelon Biosciences Inc.); mouse anti-EEA1 (610456, BD Bioscience); rabbit anti-RFP (600–401-379, ROCKLAND); sheep anti-LRP2 (gift from P. Verroust and R. Kozyraki, INSERM, Paris, France); mouse anti Flotillin-1(610821, BD Bioscience); mouse anti-α-tubulin (T5168, Sigma-Aldrich); rabbit anti-GAPDH (2118, Cell Signaling Technology); rat anti-LAMP1 (sc-19992, Santa Cruz Biotechnology); goat anti-Cathepsin-D (Cts-D; sc-6486, Santa Cruz Biotechnology); rabbit anti-EGFR (1005 sc-03, Santa Cruz Biotechnology); Alexa-488 Phalloidin (F-actin, A12379, Thermofisher Scientific); mouse anti-Transferrin Receptor Antibody (H68,4, ThermoFisher Scientific); WGA FITC Conjugate (L 4895, Sigma-Aldrich); mouse anti-Na + /K + -ATPase subunit α1 (C464.6 EMD Millipore); rabbit anti-MPR and rabbit anti-OCRL (gift from A.

Techniques: Derivative Assay, Confocal Microscopy, Two Tailed Test

Altered lysosomal dynamics and degradative capacity in Ocrl Y/− mPTCs. ( A ) Ocrl mPTCs were immunostained with anti-PI(4,5)P 2 (green) and anti-LAMP1 (red, lysosomes) and the number of PI(4,5)P 2 /LAMP1 + structures were quantified by confocal microscopy (percentage of total PI(4,5)P 2 + vesicles: n = 3 randomly selected fields per condition, each containing approximately 40–50 cells). ( B ) Representative confocal micrographs of Ocrl mPTCs immunostained with anti-LAMP1 (green). Quantification of the average LAMP1 + vesicles diameter (top, n = 4 Ocrl Y/+ and n = 6 Ocrl Y/− randomly selected fields per condition, each containing approximately 50–60 cells) and number of structures (bottom, n ≈ 200–220 cells pooled from 3 Ocrl kidneys per group, each point representing the number of LAMP1 + structure in a cell). ( C ) Ocrl mPTCs were loaded with DQ Red BSA (red, 10 μg ml −1 for 1 h at 37°C), immunostained with anti-LAMP1 (green, lysosomes) fixed and analyzed by confocal microscopy. Quantification of number of DQ Red BSA/LAMP1 + structures (percentage of total LAMP1 + structures: n = 8 randomly selected fields per condition, with each containing approximately 10–15 cells). Insets: high magnification of DQ Red BSA/LAMP1 + vesicles. ( D ) Ocrl mPTCs were serum starved for 24 h and then stimulated with EGF (100 ng ml -1 ) for the indicated times. EGFR protein levels were evaluated by western blotting and quantified relative to time 0 (starved cells; n = 3 mice per group; two-tailed unpaired Student’s t -test, * P < 0.05, ** P < 0.01 relative to Ocrl Y/+ or Ocrl Y/− starved mPTCs. ns: not significant). ( E ) Western blotting and densitometry analyses of Cathepsin D (Cts-D) protein levels in Ocrl mPTCs (n = 4 mice per group). ( F ) Ocrl mPTCs were loaded with Bodipy-FL-PepA (1 μ m , for 1 h at 37°C, green), immunostained with anti-LAMP1 antibody (red) and analyzed by confocal microscopy. Quantification of numbers of PepA/LAMP1 + structures (percentage of total LAMP1 + structures: n = 4 randomly selected fields per condition, with each containing approximately 20–25 cells). ( G ) Representative confocal micrographs showing Cy5-labeled β-lactoglobulin (red) after 120 min from tail vein injections and quantifications of the corresponding fluorescent signal in LTL + PTs from Ocrl mouse kidneys (n = 50 Ocrl PTs; each dot representing fluorescence intensity in one PT; fluorescence intensity was normalized on tubule area). Nuclei counterstained with DAPI (blue) in (A–C), (F) and (G). Scale bars: 15 μm in (A–C), 10 μm in (F) and 50 μm in (G). Plotted data represent mean ± SEM. Two-tailed unpaired Student’s t -test. ** P < 0.01, *** P < 0.001 relative to Ocrl Y/+ mPTCs or kidneys.

Journal: Human Molecular Genetics

Article Title: OCRL deficiency impairs endolysosomal function in a humanized mouse model for Lowe syndrome and Dent disease

doi: 10.1093/hmg/ddy449

Figure Lengend Snippet: Altered lysosomal dynamics and degradative capacity in Ocrl Y/− mPTCs. ( A ) Ocrl mPTCs were immunostained with anti-PI(4,5)P 2 (green) and anti-LAMP1 (red, lysosomes) and the number of PI(4,5)P 2 /LAMP1 + structures were quantified by confocal microscopy (percentage of total PI(4,5)P 2 + vesicles: n = 3 randomly selected fields per condition, each containing approximately 40–50 cells). ( B ) Representative confocal micrographs of Ocrl mPTCs immunostained with anti-LAMP1 (green). Quantification of the average LAMP1 + vesicles diameter (top, n = 4 Ocrl Y/+ and n = 6 Ocrl Y/− randomly selected fields per condition, each containing approximately 50–60 cells) and number of structures (bottom, n ≈ 200–220 cells pooled from 3 Ocrl kidneys per group, each point representing the number of LAMP1 + structure in a cell). ( C ) Ocrl mPTCs were loaded with DQ Red BSA (red, 10 μg ml −1 for 1 h at 37°C), immunostained with anti-LAMP1 (green, lysosomes) fixed and analyzed by confocal microscopy. Quantification of number of DQ Red BSA/LAMP1 + structures (percentage of total LAMP1 + structures: n = 8 randomly selected fields per condition, with each containing approximately 10–15 cells). Insets: high magnification of DQ Red BSA/LAMP1 + vesicles. ( D ) Ocrl mPTCs were serum starved for 24 h and then stimulated with EGF (100 ng ml -1 ) for the indicated times. EGFR protein levels were evaluated by western blotting and quantified relative to time 0 (starved cells; n = 3 mice per group; two-tailed unpaired Student’s t -test, * P < 0.05, ** P < 0.01 relative to Ocrl Y/+ or Ocrl Y/− starved mPTCs. ns: not significant). ( E ) Western blotting and densitometry analyses of Cathepsin D (Cts-D) protein levels in Ocrl mPTCs (n = 4 mice per group). ( F ) Ocrl mPTCs were loaded with Bodipy-FL-PepA (1 μ m , for 1 h at 37°C, green), immunostained with anti-LAMP1 antibody (red) and analyzed by confocal microscopy. Quantification of numbers of PepA/LAMP1 + structures (percentage of total LAMP1 + structures: n = 4 randomly selected fields per condition, with each containing approximately 20–25 cells). ( G ) Representative confocal micrographs showing Cy5-labeled β-lactoglobulin (red) after 120 min from tail vein injections and quantifications of the corresponding fluorescent signal in LTL + PTs from Ocrl mouse kidneys (n = 50 Ocrl PTs; each dot representing fluorescence intensity in one PT; fluorescence intensity was normalized on tubule area). Nuclei counterstained with DAPI (blue) in (A–C), (F) and (G). Scale bars: 15 μm in (A–C), 10 μm in (F) and 50 μm in (G). Plotted data represent mean ± SEM. Two-tailed unpaired Student’s t -test. ** P < 0.01, *** P < 0.001 relative to Ocrl Y/+ mPTCs or kidneys.

Techniques: Confocal Microscopy, Western Blot, Two Tailed Test, Labeling, Fluorescence

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