A protocol adapted from the SUnSET method was used to label newly synthetised proteins.35 (link),97 ,103 (link) Briefly, 400 μm-thick coronal striatal slices of the brain of 3- to 4-month-old Fmr1 KO and control mice were prepared using a vibratome. Slices were allowed to recover in aCSF at 32°C for 1 h and subsequently treated with puromycin (Cat# P8833, Sigma-Aldrich, 5 μg/mL) for 45 min. For slices subjected to pharmacological pretreatment, anisomycin (Cat# 1290, Tocris, 20 μM) and 4EGI-1 (100 μM) were added to aCSF 30 min prior to puromycin treatment. Newly synthesized proteins were end-labeled with puromycin. Striatum was micro-dissected from the brain slices and flash frozen on dry ice and lysed. 40 μg of puromycylated protein lysates were analyzed on western blotting. Protein synthesis levels were determined by taking total lane density in the molecular weight range of 10–250 kDa. Comparisons of protein synthesis levels between both genotypes were made by normalizing to the average WT signal.
Anisomycin
Manufactured by Bio-Techne
Sourced in United Kingdom
Anisomycin is a laboratory product manufactured by Bio-Techne. It is a protein synthesis inhibitor, which functions by blocking the peptidyl transferase activity of the 60S ribosomal subunit, thereby inhibiting translation elongation.
Automatically generated - may contain errors
Lab products found in correlation
Sp600125, by Bio-Techne (3 mentions)
Rapamycin, by Bio-Techne (2 mentions)
Actinomycin d, by Bio-Techne (2 mentions)
Anisomycin, by Merck Group (2 mentions)
Puromycin, by Bio-Techne (1 mentions)
Puromycin, by Merck Group (1 mentions)
Kn 93, by Merck Group (1 mentions)
Kn 93, by Bio-Techne (1 mentions)
Pd168077, by Merck Group (1 mentions)
L745870, by Merck Group (1 mentions)
Ro 10 5824, by Bio-Techne (1 mentions)
Nimodipine, by Bio-Techne (1 mentions)
Pka inhibitor fragment 6 22 amide, by Bio-Techne (1 mentions)
Mk 801, by Bio-Techne (1 mentions)
D ap5, by Bio-Techne (1 mentions)
Dopamine hydrochloride, by Merck Group (1 mentions)
Gallein, by Bio-Techne (1 mentions)
Jzl184, by Bio-Techne (1 mentions)
Am251, by Bio-Techne (1 mentions)
L name, by Bio-Techne (1 mentions)
21 protocols using anisomycin
1
Quantifying Protein Synthesis in Mouse Brain
2
Pharmacological Modulation of D4R in Synaptic Transmission
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The D4R antagonist L-745, 870 (Sigma) was first dissolved in dimethyl sulfoxide (DMSO) to make a stock solution and stored at 4 °C. This stock solution was then diluted in ACSF to a final concentration of 50 nM, ensuring that the final DMSO concentration did not exceed 0.1%. These concentrations were used as they did not affect basal synaptic transmission33 (link). The D4R agonists, PD 168077 (Sigma) and Ro-10-5824 (Tocris), were both used at 0.1 µM and anisomycin (Sigma), a protein synthesis inhibitor, and KN93 (Sigma), a CaMKII inhibitor were used at a concentration of 25 µM and 1 µM respectively. PD 168077 (Sigma), Ro-10-5824 (Tocris), anisomycin and KN93 were dissolved in DMSO to make stock solutions and later were dissolved in ACSF. The NMDA antagonist, AP5 (Sigma) was dissolved in water and was used at 50 µM concentration.
3
Murine Lung Cell Culture and Stimulation
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Primary murine lung cells were maintained at 37°C with 5% CO2. We noticed that JKP cells maintained in culture for a prolonged period of time (passage > 15) lose their growth advantage over KP cells. Therefore, all experiments were performed using cells from passages 3–10. Where indicated, cells were treated with anisomycin (MilliporeSigma, A9789, final concentration 200 ng/mL) for 30 minutes to stimulate JNK-dependent phosphorylation of c-Jun/JunD, and/or with JNK inhibitor (Tocris Bioscience, Bio-Techne; SP600125; final concentration 20 μM) for 30 minutes before harvesting (after anisomycin stimulation, if given).
Human LADC cell lines (NCI-H23, NCI-H441, NCI-H1792, and A549) were provided by the Francis Crick Institute Cell Services and cultured in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 2mM glutamine. All cells were tested Mycoplasma negative and maintained at 37°C with 5% CO2.
Human LADC cell lines (NCI-H23, NCI-H441, NCI-H1792, and A549) were provided by the Francis Crick Institute Cell Services and cultured in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 2mM glutamine. All cells were tested Mycoplasma negative and maintained at 37°C with 5% CO2.
4
Pharmacological Modulation of Synaptic Plasticity
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Drugs were bath-applied to the whole slice through the perfusion system by dilution of concentrated stock solutions (prepared in water or DMSO) in ACSF, or by adding the drugs to the patch pipette solution when it was applied intracellularly to the postsynaptic cell only. If the drug was not water-soluble, vehicle control experiments were carried out. For each set of recordings, interleaved control and drug conditions were carried out and were pseudorandomly chosen. The following drugs were used in this study: 100 μM dopamine hydrochloride (Sigma–Aldrich, Dorset, United Kingdom), 100 μM D-AP5 (Tocris Bioscience, Bristol, United Kingdom), 10 µM nimodipine (Tocris Bioscience), 1 µM PKA inhibitor fragment (6-22) amide (Tocris Bioscience), 0.5 mM anisomycin (stock solution in EtOH; Tocris Bioscience), and 1 mM MK801 (Tocris Bioscience).
5
Pharmacological Modulation of Signaling Pathways
6
Investigating JNK-BAD Signaling Pathway
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Roswell Park Memorial Institute 1640 (RPMI-1640) and high glucose Dulbecco’s modified Eagle’s (DMEM) medium were purchased from Hyclone (South Logan, UT, USA). Fetal bovine serum (FBS) was purchased from Gibco (CA, USA). JNK agonist anisomycin was purchased from Tocris biosciences (Bristol, UK). Anti- SH3BP5 (#11127-2-AP), Caspase 3 (#19677-1-AP), JNK (#24164-1-AP), BAD (#10435-1-AP), and Actin (#60008-1-Ig) antibodies were purchased from Proteintech Group (Chicago, IL, USA). Anti- p-JNK Thr183/Tyr185 (#9255) and p-BAD Ser112 (#5284) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).
7
Quail Lens Cell Culture and Inhibitor Treatment
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Primary lens cell cultures were prepared as described previously47 (link) using lenses isolated from E9 quail lenses. Briefly, cells were isolated by trypsinization and agitation and plated on laminin, and cultured in Complete Medium. After the cells had differentiated and formed mature lentoid structures (D10) they were treated for 24 h with MAPK/JNK inhibitor, SP600125 (25 µM), the MTOR inhibitor, rapamycin (100 nM), the MAPK/JNK activator anisomycin (Tocris Bioscience, 1290; 0.5 μM) or the vehicle control DMSO. Details on preparation of the SP600125 inhibitor have been previously described.66 (link) For treatment of embryonic lenses with inhibitors in ex vivo organ culture or in primary lens cell cultures complete medium was replaced with medium containing SP600125 (25 μM), JNK-IN-8 (1 μM), rapamycin (100 nM) or anisomycin (0.5 μM).
8
Histone Acetylation Regulation of aPKC
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Histone deacetylase inhibitors trichostatin A (TSA, final concentration 100 nM, Sigma) and sodium butyrate (NaB, final concentration 5 mM, Sigma-Aldrich) were used for investigation of the role of histone acetylation in the regulation of aPKC expression. Cultures were incubated with TSA for 4, 8, 19, 48 hours, or with NaB for 19 hours.
Transcription inhibitor actinomycin D (ActD, final concentration 200 nM, 4 μM; Sigma-Aldrich), and protein synthesis inhibitor anisomycin (Ani, final concentration 10 μM, 100 μM; Tocris) were used for verification of experiments with histone deacetylase inhibitors. Experimental cultures were pretreated with ActD or Ani for 1 hour (without washout), followed by continuous incubation with TSA (19 hours). Control cultures were incubated with ActD or Ani for 20 hours.
Transcription inhibitor actinomycin D (ActD, final concentration 200 nM, 4 μM; Sigma-Aldrich), and protein synthesis inhibitor anisomycin (Ani, final concentration 10 μM, 100 μM; Tocris) were used for verification of experiments with histone deacetylase inhibitors. Experimental cultures were pretreated with ActD or Ani for 1 hour (without washout), followed by continuous incubation with TSA (19 hours). Control cultures were incubated with ActD or Ani for 20 hours.
9
Immunoblotting and Immunocytochemistry Protocols
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Alexa Fluor 488-coupled goat anti-rabbit, Alexa Fluor 568-coupled goat anti-mouse, and anti-rabbit phospho-eIF2αS52 antibodies were from Invitrogen. Rabbit anti-PKR-like endoplasmic reticulum kinase (PERK) antibody was from Rockland. Mouse anti-eIF2α (L57A5), and rabbit anti-PERK (C33E10) and rabbit anti-mGluR1 antibodies were from Cell Signaling Technology. Anti-puromycin (12D10) antibody was kindly provided by Dr. P. Pierre. Anti-mGluR5 antibody was from R&D Systems. 3,3′-Diaminobenzidine (DAB) substrate staining kit was from Vector Laboratories. (RS)-3,5-Dihydroxyphenylglycine (DHPG) and anisomycin were obtained from Tocris Bioscience. Puromycin (P8833) and mouse anti-actin and anti-MAP2 antibodies were from Sigma.
10
Neurotransmitter Signaling Pathway Modulation
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4-Aminopyridine, PTX, poly-L-lysine, D(−)-2-amino-5-phosphonopentanoic acid, Trolox, DAPI nuclear dye, Nocodazole, and Ciliobrevin D were purchased from Sigma. Bicuculline methiodide, MNI-caged-L-glutamate, Anisomycin were purchased from Tocris Bioscience. Leptomycin B (10 nM) was purchased from Beyotime. Peptides used are Tat-APPL113 (YGRKKRRQRRRRRASEKQKEIERVKEK) and Tat-APPL1Scr (YGRKKRRQRRRRRASEKQKEIEAAAAA). The following antibodies were used: anti-APPL1 (sc-67402) and anti-Rab5 (sc-46692) from Santa Cruz Biotechnology, anti-pERK (4370S) and anti-GAPDH (2118) from Cell Signaling Techonology, anti-HDAC2 (ab32117), anti-Importin α1 (ab84440), anti-Histone H4K5 (ab51997), anti-Histone H4K12 (ab177793), anti-APPL2 (ab95196), and anti-Histone H4 (ab10158) from Abcam, anti-MAP2 (M9942, M3696), anti-FLAG (F1804), and anti-APPL1 (1409089) from Sigma–Aldrich, and anti-pCREB (06-519 and 04-218) from Millipore. Glutathione sepharose beads and protein A sepharose beads were purchased from GE Healthcare. Phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitor cocktails 2 and 3 were purchased from Sigma. Horseradish peroxidase (HRP)-linked goat anti-mouse immunoglobulin G (IgG), goat anti-rabbit IgG, and donkey anti-goat IgG, secondary antibodies conjugated to Dylight (488 or 555), and chemiluminescence kit were purchased from Pierce.
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