The PI4 kinase inhibitors GSK-A1 (SYNkinase, Parkville, Australia) and phenylarsine oxide (PAO; Sigma-Aldrich, St Louis, USA) were dissolved in 1 mM (GSK-A1) and 100 mM (PAO) stock solutions in DMSO and diluted to their working conditions (100 nM GSK-A1; 30 mM PAO) in extracellular solution. Cells were incubated in PI4 kinase inhibitors for 10 min (GSK-A1) and 30 min (PAO) prior to imaging. Rapamycin was purchased as 5 mM solution in DMSO (Calbiochem, San Diego, USA) and diluted to a 1 µM working solution in extracellular solution (5.8 mM KCl, 144 mM NaCl, 0.9 mM MgCl2, 1.3 mM CaCl2, 0.7 mM NaH2PO4, 5.6 mM D-glucose, 10 mM HEPES pH 7.4).
Gsk a1
Manufactured by Merck Group
Sourced in United States
GSK-A1 is a laboratory equipment product developed by Merck Group. It is designed for specific tasks within a controlled environment. The core function of GSK-A1 is to enable precise and consistent measurements or analyses. No further details on its intended use can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Trimethylsilyldiazomethane, by Merck Group (1 mentions)
L glutamate, by Merck Group (1 mentions)
Oxotremorine methiodide oxo m, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Sodium formate, by Merck Group (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Wortmannin, by Merck Group (1 mentions)
Texas red 1 2 dihexadecanoyl sn glycero 3 phosphoethanolamine triethylammonium salt texas red dhpe, by Thermo Fisher Scientific (1 mentions)
Brain phosphatidylserine, by Avanti Polar Lipids (1 mentions)
Egg phosphatidylcholine, by Avanti Polar Lipids (1 mentions)
1 2 dioleoyl sn glycero 3 phospho 1 myo inositol 3 phosphate, by Avanti Polar Lipids (1 mentions)
Sir tubulin, by Spirochrome (1 mentions)
Rapamycin, by Thermo Fisher Scientific (1 mentions)
Motorized tirf illuminator, by Nikon (1 mentions)
Tie inverted microscope stand, by Nikon (1 mentions)
5.5 scmos camera, by Oxford Instruments (1 mentions)
A11011, by Thermo Fisher Scientific (1 mentions)
Fluorescent phalloidin, by Thermo Fisher Scientific (1 mentions)
Anti sheep red blood cell antibodies, by Cedarlane (1 mentions)
7 protocols using gsk a1
1
PI4 Kinase Inhibitors for Imaging
2
Oxo-M Calcium Imaging with PI4 Kinase Inhibitors
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Oxotremorin-M (Oxo-M, Tocris Bioscience, Bristol, UK) was prepared in a 10mM stock solution in water and diluted to 10 µM in extracellular solution. The PI4 kinase inhibitors GSK-A1 (SYNkinase, Parkville, Australia) and phenylarsine oxide (PAO, Sigma Aldrich, St Louis, US)
were dissolved in 1 mM (GSK-A1) and 100 mM (PAO) stock solutions in DMSO and diluted to their working conditions (10 nM and 100 nM GSK-A1; 30 mM PAO) in extracellular solution.
Cells were incubated in PI4 kinase inhibitors for 10 min (GSK-A1) and 30 min (PAO) prior to imaging.
were dissolved in 1 mM (GSK-A1) and 100 mM (PAO) stock solutions in DMSO and diluted to their working conditions (10 nM and 100 nM GSK-A1; 30 mM PAO) in extracellular solution.
Cells were incubated in PI4 kinase inhibitors for 10 min (GSK-A1) and 30 min (PAO) prior to imaging.
3
Glutamate Signaling Pathway Analysis
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L-glutamate, oxotremorine methiodide (oxo-M), GSK-A1, sodium formate, HCl, and trimethylsilyl-diazomethane (2.0 M in diethyl ether or hexanes) were from Sigma-Aldrich. Mass spectrometry-grade methanol, chloroform, dichloromethane, and acetonitrile were from Thermo Fisher Scientific.
4
Fluorescent Labeling Reagents for Cell Imaging
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AF-488 (1:500) labelled-Tf and AF-647 labelled WGA (1:200) were from Invitrogen (Thermo Fisher Scientific); siR-Tubulin (1:1,000) was from Spirochrome. PAO (PhenylArsineOxide), wortmannin, GSK-A1, and rapamycin were from Sigma Aldrich, and stock solutions were dissolved in dimethyl sulfoxide (DMSO; Sigma Aldrich). The following reagents for in vitro studies were from Avanti Polar Lipids: Egg phosphatidylcholine, brain phosphatidylserine, and 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-3'-phosphate) 18:1 (EPC, bPS, and PI3P, respectively; in chloroform); brain phosphatidylinositol-4-phosphate (bPI4P; in chloroform/methanol/water [20:9:1]); brain phosphatidylinositol-4,5-bisphosphate, C24:1 Galactosyl(β) Ceramide, and 1,2-dioleoyl-sn-glycero-3-phospho-l -serine (bPI[4,5]P2, GalCer, and DOPS; in chloroform/methanol [90/10% v/v]). Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (Texas Red DHPE) and BODIPY Texas Red ceramide (BODIPY TR ceramide) was from Thermo Fisher Scientific.
5
TIRF Microscopy Imaging of Rapamycin and GSK-A1
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A Nikon motorized TIRF illuminator mounted on a Nikon TiE inverted microscope stand was used. Laser excitation was fiber coupled from a four-line (405, 488, 561 and 638 nm) combiner (Oxxius).Emission was collected through dual pass filters from Chroma: blue/yellow-orange (420–480 nm / 570–620 nm) and green/far-red (505–550 nm / 650–850 nm). Imaging was performed through a 1.45 NA, plan apochromatic oil immersion objective (Nikon) using an Andor Zyla 5.5 sCMOS camera. For time-lapse imaging, up to 10 individual fields were marked using the motorized stage and imaged every 30 s. 0.5 ml of 5 µM Rapamycin (Fisher Scientific BP2963–1) + 150 nM GSK-A1 (Sigma SML2453) were added after 2 min to achieve bath concentrations of 1 µM and 30 nM, respectively.
6
Immunoblotting and Microscopy Protocols
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The following antibodies were used in this study: mouse anti-Lamp1 (DHSB, H4A3, dilution: 1:1000), rabbit anti-ORP1 (Abcam, ab131165, dilution: 1:1000), mouse anti-Drp1 (BD Biosciences, 611112, dilution: 1:1000), rabbit anti-VAPA (Novus Biologicals, NBP1-31237, dilution: 1:1000), rabbit-anti-VAPB (MilliporeSigma, HPA013144, dilution: 1:1000), mouse-anti GAPDH HRP conjugated (Novus Biologicals, NB300-328H, dilution: 1:10000), goat anti-Rabbit HRP (ThermoFisher, 31460, dilution: 1:10000), goat anti-Mouse HRP (Cedarlane, CLCC30007, dilution: 1:10000) and goat anti-Mouse Alexa 568 (ThermoFisher, A-11011, dilution: 1:1000). GA3-AM (SML1959), Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (C2759), and GSK-A1 (SML2453) were purchased from MilliporeSigma. MitoTracker™ Deep Red FM - Special Packaging (M22426) was purchased from ThermoFisher.
7
Immunofluorescence assay for Lpd and VASP
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Rabbit polyclonal Lpd antibody (Krause et al., 2004 (link)) was used for immunofluorescence at 1:200. Rabbit monoclonal VASP (9A2) antibody (#3132; Cell Signaling Technology) was used for immunofluorescence at 1:200. SRBCs 10% suspension were purchased from MP Biomedicals. Anti-sheep red blood cell antibodies were purchased from Cedarlane Laboratories and supplied by MP Biomedicals (Cat #0855806). Fluorescent secondary antibodies against mouse (#711-165-150) and rabbit (#711-605-152, 711-545-152, and 711-156-152) were purchased from Jackson ImmunoResearch Labs. Paraformaldehyde (16% wt/vol) was purchased from Electron Microscopy Sciences. Additional reagents: Wortmannin (681675; Sigma-Aldrich), PI-103 (528100; Sigma-Aldrich), GDC-0941 (509226; Millipore Sigma), GSK-A1 (kindly provided by Dr. Tamas Balla, National Institutes of Health, Bethesda, MD [Bojjireddy et al., 2014 (link)]), AS1949490, and K118 (Echelon Biosciences), fluorescent phalloidin (Molecular Probes), PBS, and HBSS (Wisent Bioproducts).
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