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2 protocols using n ethylmaleimide

1

Antibody and Reagent Sources for Cell Signaling

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The following antibodies were used in this study: anti-USP33, anti-slug, and anti-twist1 antibodies were purchased from ProteinTech Group (Chicago, IL, USA); anti-CXCR4 antibody was purchased from BD Bioscience (Franklin Lakes, NJ, USA); anti-HA and anti-Flag M1 antibodies were from Sigma-Aldrich (St. Louis, MO, United States); anti-ppERK, anti-ERK, and anti-β-arrestin2 antibodies were from Cell Signaling Technology (Boston, MA, USA); anti-E-cadherin, anti-N-cadherin, anti-snai1, and anti-β-actin antibodies were purchased from Santa Cruz (Dallas, TX, USA).
N-ethylmaleimide (NEM, deubiquitinase inhibitor), SDF-1 (SDF-1α, CXCR4 agonist), AMD3100 octahydrochloride hydrate (1,1′-[1,4-Phenylenebis(methylene)]bis-1,4,8,11-tetraazacyclotetradecane octahydrochloride, CXCR4 antagonist), dynasore hydrate (3-Hydroxy-naphthalene-2-carboxylic acid (3,4-dihydroxy-benzylidene)-hydrazide hydrate, dynamin inhibitor), HA affinity beads, and Flag affinity beads were all purchased from Sigma-Aldrich.
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2

Neuronal Culture Preparation and Protein Manipulation

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Neurobasal-A medium (no glucose, no sodium pyruvate), B-27 supplement and B-27 supplement minus antioxidants were purchased from Life Technologies (Grand Island, NY). Clasto-lactacystin β-lactone (CLβL) and MG132 were purchased from Cayman Chemical (Ann Arbor, Michigan). Deubiquitinase inhibitor bAP15 was purchased from Ubiquitin-Proteasome Biotechnologies (Aurora, CO). CGP3466B was purchased from Tocris Bioscience (Bristol, United Kingdom). Nω-nitro-l-arginine methyl ester (L-NAME), 7-nitroindazole, protein G plus-agarose and n-ethylmaleimide were purchased from Santa Cruz Biotechnology (Paso Robles, CA). Ketamine hydrochloride was purchased from Spectrum Chemicals & Laboratory Products (Gardena, CA). N-methyl-d-aspartic acid (NMDA) and Siah1 shRNA (verified) lentiviral plasmid were purchased from Sigma-Aldrich (St. Louis, MO). Rapamycin was purchased from Cell Signaling Technology (Danvers, MA).
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