Cultured hippocampal slices were transferred to a submersion-type holding chamber containing aCSF gassed with 5% CO2/95% O2 at 29 °C for 30 min before the induction of plasticity protocols. To induce LTP chemically, a cocktail containing 50 µM forskolin, 0.1 µM rolipram, and 100 µM PTX was prepared in a Mg-free aCSF and incubated for 15 min (cLTP); or a tetraethylammonium (TEA, 25 mM) solution was incubated for 10 min. For induction of LTD, the mGluR1/5 agonist DHPG (100 µM, 10 min) was used. After washing out the drugs, slices were homogenized in lysis buffer (150 mM NaCl, 50 mM Tris–HCl pH 7.4, 1% Triton X-100) containing protease and phosphatase inhibitor cocktails (complete mini EDTA-free and phosSTOP, Roche), and processed for western blot, as previously described (Briz and Baudry 2014 ). Hippocampal samples from adult rats following perinatal exposure to pesticides were similarly processed for western blotting. See Supplementary Information for details on procedures and antibodies used.
Complete mini edta free and phosstop
Manufactured by Roche
Complete mini EDTA-free and PhosSTOP is a laboratory product that consists of a cocktail of phosphatase inhibitors. It is designed to prevent the dephosphorylation of proteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
0.2 μm nitrocellulose membrane, by Bio-Rad (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid assay, by Santa Cruz Biotechnology (1 mentions)
Polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
Immunocruz western blotting luminol reagent, by Santa Cruz Biotechnology (1 mentions)
Fusion fx7 imaging system, by Vilber (1 mentions)
Quick western kit, by LI COR (1 mentions)
Protein a g plus sepharose beads, by Santa Cruz Biotechnology (1 mentions)
Gradient gel, by Thermo Fisher Scientific (1 mentions)
Odyssey infrared scanner, by LI COR (1 mentions)
Image studio software, by LI COR (1 mentions)
Odyssey software, by LI COR (1 mentions)
Catalog no a5441, by Merck Group (1 mentions)
Gradient tris glycine gel, by Thermo Fisher Scientific (1 mentions)
7 protocols using complete mini edta free and phosstop
1
Electrophysiological and Biochemical Analysis of Hippocampal LTP and LTD
2
Chronic Haloperidol Decanoate in Rats
3
Subcellular Fractionation of Hippocampal Slices
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Subcellular fractions were prepared as previously described (Gardoni et al., 2009 (link)). All centrifugation steps were performed at 4°C, and buffers were maintained on ice. All buffers contained protease and phosphatase inhibitor cocktails (Complete Mini EDTA-free and PhosSTOP; Roche). Briefly, hippocampal slices were homogenized on a glass-Teflon potter (25 strokes) with buffer containing 0.32 M sucrose, 1 mM Hepes, 1 mM MgCl2, 1 mM NaHCO3, and 1 mM EDTA. Nuclei and myelin were removed by 10-min centrifugation at 10,000 g. Subsequently, the supernatant was centrifuged at 13,000 g for 15 min to yield the crude synaptosomal fraction (pellet) and the cytosol and light membrane/microsome fraction (supernatant). After washes with the homogenization buffer, the pellet was resuspended in a buffer containing 75 mM KCl and 1% Triton X-100. Both the resuspended pellet and supernatant were centrifuged for 1.5 h at 100,000 g. From the centrifuged supernatant, we obtained the cytosolic fraction and a pellet corresponding to the microsomal fraction. The centrifuged crude synaptosomal fraction yielded a supernatant corresponding to the Triton-soluble fraction (presynaptic and extrasynaptic fraction) and a pellet enriched in the postsynaptic density. Both pellets were washed extensively and resuspended in 0.1% NP-40 and 20 mM Hepes for western analysis.
4
Whole-Cell Protein Lysate Preparation and Western Blot Analysis
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Whole-cell protein lysate preparation was performed with RIPA buffer (15 mM HEPES, 150 mM NaCl, 10 mM EGTA, 2% Triton X-100) supplemented with protease and phosphatase inhibitors (complete mini EDTA-free and PhosSTOP, Roche) as described before [14 (link),21 (link),35 ]. Bicinchoninic acid assay (Santa Cruz Biotechnology, Texas, USA) was used for protein quantitation according to the manufacturer's protocol. Laemmli buffer was mixed with cell lysates, denatured for 5 min at 95 °C and loaded in a 10% polyacrylamide gel (Thermo Fisher Scientific). Lysates were separated by electrophoresis at 120 V and transferred to a PVDF membrane. Membranes were blocked in 5% nonfat dry milk in TBS-T and incubated in primary antibody in 5% nonfat dry milk in TBS-T overnight at 4 °C. Antibodies and dilutions are listed in Supplementary Table 1 . Membranes were washed three times and incubated with the secondary antibody. Proteins were visualized with ImmunoCruz Western Blotting Luminol Reagent (Santa Cruz Biotechnology) and the Fusion FX7 imaging system was used for detection (Vilber Lourmat).
5
Western Blot Analysis of Cell Lysates
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Cells were harvested, lysed, and sonicated in low salt lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, pH 7.4) supplemented with protease and phosphatase inhibitors (Complete mini EDTA-free and PhosSTOP, Roche Applied Science). Total protein was concentrated down using trichloroacetic acid precipitation and reconstituted in 2X Laemmli buffer. Samples were loaded onto 15% Tris-glycine gels or 4–20% precast Tris-glycine gradient gels (Life Technologies), subjected to SDS-PAGE, and transferred onto 0.2 μm nitrocellulose membranes (BioRad). Membranes were blocked in Odyssey Blocking Buffer for 1 h and immunoblotting was performed using the monoclonal antibodies listed above at 1:1000 dilution, incubated overnight at 4 °C. Blots were washed three times in PBS-T and incubated in donkey anti-rabbit 800 or donkey anti-mouse 680 fluorescent secondary antibodies (Li-Cor) for one hour at room temperature. Protein bands were visualized using a Li-Cor Odyssey Infrared Imaging System (Li-Cor).
6
Immunoprecipitation and Western Blotting
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Cells were collected in lysis buffer (1 mM EGTA, 1 mM EDTA, 10 mM Tris [pH 7.4], 150 mM sodium chloride, 1% deoxycholate, 1% NP-40, and supplemented with protease and phosphatase inhibitor cocktail tablets [cOmplete mini EDTA-free and PhosStop; Roche]). Lysates were precleared with 10 μl of protein A/G PLUS Sepharose Beads (Santa Cruz Biotechnology) for 1 h at 4°C. The precleared lysates were then incubated with 20 μl of protein A/G PLUS Sepharose Beads, which had previously been coupled with the appropriate primary antibody for 2 h at 4°C on an orbital shaker. The beads were washed three times with lysis buffer supplemented with protease and phosphatase inhibitors. The immunoprecipitation complexes were eluted from the beads by boiling in 2× SDS buffer for 5 min.
For Western blotting analyses, protein lysates/coimmunoprecipitation complexes were resolved on a 4%–12% gradient gel (Invitrogen) and blotted on a nitrocellulose membrane with the appropriate primary antibodies (1:1,000) and IRDye-conjugated anti-mouse, anti-goat, or anti-rabbit secondary antibodies or the Quick Western Kit (926-69100; LI-COR), as appropriate. Images were acquired on a LI-COR Odyssey infrared scanner. Densitometric quantification of bands was performed using Image Studio software (LI-COR).
For Western blotting analyses, protein lysates/coimmunoprecipitation complexes were resolved on a 4%–12% gradient gel (Invitrogen) and blotted on a nitrocellulose membrane with the appropriate primary antibodies (1:1,000) and IRDye-conjugated anti-mouse, anti-goat, or anti-rabbit secondary antibodies or the Quick Western Kit (926-69100; LI-COR), as appropriate. Images were acquired on a LI-COR Odyssey infrared scanner. Densitometric quantification of bands was performed using Image Studio software (LI-COR).
7
Quantitative Western Blot Analysis
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U87MG and GL261 cells were harvested and lysed in low salt lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, pH 7.4) supplemented with protease and phosphatase inhibitors (Complete mini EDTA-free and PhosSTOP, Roche Applied Science). Normalized samples were loaded onto 4–12% precast Tris-glycine gradient gels (Life Technologies), subjected to SDS-PAGE, and transferred onto 0.2 μm nitrocellulose membranes (BioRad). Immunoblotting was performed using monoclonal antibodies against transferrin receptor (ThermoFisher, Catalog No. 13-6800) and actin (Sigma, Catalog No. A5441) each at 1:1000 dilution. Protein bands were visualized following incubation with Li-Cor fluorescent secondary antibodies (Li-Cor, Catalog No. P/N 925-32212 and P/N 925-68072) at 1:20,000 dilution using a Li-Cor Odyssey Infrared Imaging System (Li-Cor) and quantified using Odyssey software (Li-Cor). Actin was used as a loading control. Protein band intensity quantified using Li-Cor Odyssey software. Intensity of bands averaged over three separate experiments. Uncropped blots are included in Supplementary Information Section .
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