Lpcat 3 has 13 exons, with a translation start codon (ATG) in the first exon. This gene is 41 kb in length. We replaced exons 2–12 (5,550 bp) with a Neo cassette and generated the “null” Lpcat 3 allele. To rescue the lethality of LPCAT3 KO male and female mice, we gavaged the newborns, starting from day 3 after birth, with PCs (P3556, sigma)/olive oil (0.5mg/ml, first week 10 μl/day, second week 20μl/day; and third week 50μl/day). At age 4-weeks old, the supplementation was stopped and the KO mice were fed chow diet same as WT animals. All mice, male or female, used in this study were 8–12 weeks old, with a C57BL/6J genetic background. The SUNY Downstate Medical Center Animal Care and Use Committee approved all animal procedures.
P3556
Manufactured by Merck Group
Sourced in Australia, United States
P3556 is a laboratory equipment product manufactured by Merck Group. It serves as a general-purpose device for various scientific applications. The core function of this product is to perform specific tasks within a controlled laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
Microplate reader, by Thermo Fisher Scientific (4 mentions)
P7769, by Merck Group (3 mentions)
Hrp conjugated goat anti human igg antibody, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated mouse anti human his antibody, by BioLegend (2 mentions)
70ti rotor, by Beckman Coulter (1 mentions)
Q500 sonicator, by Qsonica (1 mentions)
By4741, by Thermo Fisher Scientific (1 mentions)
Fluorchem 5500 imaging station, by Bio-Techne (1 mentions)
P7693, by Merck Group (1 mentions)
Avanti mini extruder, by Avanti Polar Lipids (1 mentions)
Sg65i, by Merck Group (1 mentions)
11 protocols using p3556
1
LPCAT3 Knockout Murine Rescue
2
Reconstitution of EfrEF Proteoliposomes
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E. coli polar lipids extracted from E. coli total lipids (Avanti lipids 100500 P) and L-α-Phosphatidylcholine (from egg yolk, Type XVI-E, ≥ 99% (TLC) P3556 Sigma) were dissolved in chloroform. Lipids were mixed in a 3:1 (w/w) ratio, chloroform was evaporated and dried lipids were dissolved in reconstitution buffer (50 mM K-HEPES pH 7.0). Lipids were sonicated to generate small unilamellar vesicles (SUVs). SUVs were flash-frozen in liquid nitrogen and thawed four times to fuse the SUVs to large multilamellar vesicles (LMVs). Large unilamellar vesicles (LUVs) were finally formed by extruding LMVs through a 400 nm polycarbonate filter. LUVs were diluted to a working concentration of 4 mg/ml and destabilized using 5.25 mM Triton X-100. Detergent-purified EfrEF variants were added to the destabilized liposomes at a protein:lipid ratio of 1:100. Detergent molecules were removed by four rounds of adding and removing 200 mg Bio-Beads (SM-2 polystyrene beads, Bio-Rad). Proteoliposomes were harvested by centrifugation (40’000 rpm, 70 Ti rotor, Beckman) and resuspended in 50 mM K-HEPES pH 7.016 (link).
3
Optimized Freezing Media Preparation
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According to physicochemical data of water samples, the optimum sample was selected to prepare the freezing media which was consisted of 1000 Hz ELEF-treated water supplemented with TES, tris, glycerol (10%), DMSO (2%) and soybean lecithin (1%; P3556, purity > 99%, Sigma). Freezing media were set at 325 mOsm and adjusted to pH 7.5, before adding lecithin. Lecithin was added to TEST buffer and the mixture was centrifuged at 1,000 g for 20 min. The supernatant was then filtered through 0.45 μm Acrodisc syringe filters (Merck Millipore Company, U.S). Finally, glycerol and DMSO were added to the TEST lecithin medium.
4
Quantifying Ebola Virus Protein Interactions
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For detection of direct interactions between Ebola virus GPs and ADI-15946, His-tagged GPs 2 μg/ml were first coated with coating buffer (0.1 M Na2CO3 and NaHCO3 [pH 9.6]) on 96-well plates overnight at 4°C. Wells were washed with PBST (PBS plus 0.2% Tween 20) and saturated for 1 h at 37°C with PBS–4% nonfat milk. Three-fold dilutions of ADI-15946 were added, and the mixture was incubated for 1 h at 37°C. Bound particles were detected with HRP-conjugated goat anti-human IgG antibody (Invitrogen; A18817). PS-associated ELISAs were carried out as previously described (57 (link)). PS (Sigma; P7769) and phosphatidylcholine (PC) (Sigma; P3556) were first dissolved in chloroform as a stock solution. Then, the solution was diluted to 5 μg/mL with methanol and added to the ELISA plate. Upon being dried, the plates were blocked with Tris-buffered saline (TBS)–4% bovine serum albumin (BSA) fraction V. TIM-1 ECD, TIM-1 IgV, TIM-1 ΔIgV, and control protein were diluted in TBS–10 mM CaCl2, and the mixture was added to the wells and incubated at 37°C for 1 h. The plates were washed with TBST (TBS plus 0.05% Tween 20) before incubation with HRP-conjugated mouse anti-human His antibody (Biolegend; 652503). The absorbance at 450 nm was measured by microplate reader (Thermo Fisher Scientific).
5
Quantifying Ebola Virus Protein Interactions
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For detection of direct interactions between Ebola virus GPs and ADI-15946, His-tagged GPs 2 μg/ml were first coated with coating buffer (0.1 M Na2CO3 and NaHCO3 [pH 9.6]) on 96-well plates overnight at 4°C. Wells were washed with PBST (PBS plus 0.2% Tween 20) and saturated for 1 h at 37°C with PBS–4% nonfat milk. Three-fold dilutions of ADI-15946 were added, and the mixture was incubated for 1 h at 37°C. Bound particles were detected with HRP-conjugated goat anti-human IgG antibody (Invitrogen; A18817). PS-associated ELISAs were carried out as previously described (57 (link)). PS (Sigma; P7769) and phosphatidylcholine (PC) (Sigma; P3556) were first dissolved in chloroform as a stock solution. Then, the solution was diluted to 5 μg/mL with methanol and added to the ELISA plate. Upon being dried, the plates were blocked with Tris-buffered saline (TBS)–4% bovine serum albumin (BSA) fraction V. TIM-1 ECD, TIM-1 IgV, TIM-1 ΔIgV, and control protein were diluted in TBS–10 mM CaCl2, and the mixture was added to the wells and incubated at 37°C for 1 h. The plates were washed with TBST (TBS plus 0.05% Tween 20) before incubation with HRP-conjugated mouse anti-human His antibody (Biolegend; 652503). The absorbance at 450 nm was measured by microplate reader (Thermo Fisher Scientific).
6
Liposome Preparation from Egg Yolk Lecithin
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An egg yolk lecithin (L -α-phosphatidylcholine [egg yolk, Type XVI-E, ≥ 99% (TLC), lyophilized powder, P3556; Sigma-Aldrich, Sydney, Australia]) stock solution of 2% (w/v) was prepared under nitrogen flow to prevent oxidation and then stored in the dark at 4°C. Fresh liposomes were prepared through probe sonication (Q500 Sonicator; QSonica, Newtown, CT) of the egg yolk lecithin solution using a 3.2 mm microtip at 30% amplitude and subjected to a pulse mode for 4 min (5 s on-off alterations).
7
Yeast Growth in Lipid Supplementation
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S. cerevisiae BY4741 (Open Biosystems) was grown in YPD medium in the absence or presence of additional phosphatidylinositol or phosphatidylcholine (200 μM each, Sigma-Aldrich 79401 or P3556, respectively). OD600 values of exponentially growing yeast cultures were recorded with a robotic system. Twelve-point serial dilutions were assayed in 96-well plates with a reaction volume of 150 μl.
8
Quantification of Phospholipids in Muscle
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Total lipids from plantaris homogenates (1.25 mg) were extracted as previously described [18 (link)] and were subsequently spotted onto high-performance thin layer chromatography plates (HPTLC; 5633–5, EMD Chemicals, Darmstadt, Germany) and individual phospholipids were separated using a chloroform:methanol:acetic acid:water (100:75:7:4) solvent system [4 (link)]. A standard curve (0.5, 1.0, 2.0, 4.0 μg) of purified PC (P3556, Sigma Aldrich, MO, USA), and a standard curve (0.2, 0.4, 0.8, 1.6 μg) of purified PE (P7943, Sigma Aldrich) were also loaded onto each HPTLC plate. After allowing the solvent to run up each plate for 45 min, the plates were then charred at 180 °C with a 10% (w/v) copper (II) sulfate in 8% phosphoric acid solution for 15 min [19 (link)]. Images of the HPTLC plates were captured using a CCD camera on a Fluorchem 5500 imaging station (Alpha Innotech, CA, USA) under reflective white light. Densitometry analyses were then performed using imageJ (National Institutes of Health, MA, USA) and the standard curve of PC and PE (Fig. 1 ) were used to calculate the absolute PC and PE concentrations (per 1.25 mg of muscle) in plantaris.![]()
Representative images of PC (
9
Lipid Extraction and Separation for Bacterial Assay
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Standard substance SLs and PLs including PC (Sigma-Aldrich; P3556), PE (Sigma-Aldrich; P7693), SM (Sigma-Aldrich; S0756), Sph (Avanti; 860490P), and TL of mouse liver were dissolved in DMSO and mixed with cultured E. coli bacteria to 1 mM final concentration before spotting plates.
The separation of SLs was performed as follows: ∼200,000 synchronized young adult N2 worms were harvested for TL extraction. TL (content 150 µg fatty acids) were loaded in TLC silica plates (Merck) and developed to the top of the plate in chloroform/methanol/water/acetic acid (65:25:4:3, vol/vol/vol/vol) for separated Glc and SM, chloroform/methanol/water (65:25:4, vol/vol/vol; Hayashi et al., 2018 (link)) for separated Sph, and chloroform:ethanol:water:triethylamine (30:35:7:35, vol/vol/vol/vol; Lee et al., 2008 (link)) for separated PC, PE, PI, and PS. Individual SM, Sph, PC, and PE bands were scraped from the TLC plates and dissolved in chloroform/methanol (1:1, vol/vol), and ultrasound was performed for 30 min. The samples were centrifuged, and the liquid was transferred to a new glass tube, immediately blown dry with nitrogen, and redissolved with chloroform. All recycled SLs and PLs were dissolved in 100% DMSO and mixed with 1 ml cultured bacteria before spotting plates.
The separation of SLs was performed as follows: ∼200,000 synchronized young adult N2 worms were harvested for TL extraction. TL (content 150 µg fatty acids) were loaded in TLC silica plates (Merck) and developed to the top of the plate in chloroform/methanol/water/acetic acid (65:25:4:3, vol/vol/vol/vol) for separated Glc and SM, chloroform/methanol/water (65:25:4, vol/vol/vol; Hayashi et al., 2018 (link)) for separated Sph, and chloroform:ethanol:water:triethylamine (30:35:7:35, vol/vol/vol/vol; Lee et al., 2008 (link)) for separated PC, PE, PI, and PS. Individual SM, Sph, PC, and PE bands were scraped from the TLC plates and dissolved in chloroform/methanol (1:1, vol/vol), and ultrasound was performed for 30 min. The samples were centrifuged, and the liquid was transferred to a new glass tube, immediately blown dry with nitrogen, and redissolved with chloroform. All recycled SLs and PLs were dissolved in 100% DMSO and mixed with 1 ml cultured bacteria before spotting plates.
10
Preparation of Lipid Vesicles
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Lipid mixture containing 40% phosphatidylcholine (Sigma-Aldrich #P3556), 20% phosphatidylserine (Sigma-Aldrich #P7769) and 40% cholesterol (Sigma-Aldrich #P8667) were prepared in chloroform and then dried using nitrogen gas. Dried lipids were rehydrated to a total lipid concentration of 1 mM in 20 mM Hepes pH 7.5, 150 mM NaCl, 3 mM NaN 3 , and 0.1 mM TCEP buffer (Buffer L). After 3 cycles of vortexing and sonication in batch sonicator large unilamellar vesicles (LUVs) were generated by extruding the sample through a polycarbonate membrane of 200 nm pore size using Avanti Mini-Extruder (Avanti Polar Lipids).
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