Journal: Journal of neurochemistry
Article Title: Inhibition of EGF-induced ERK/MAP kinase-mediated astrocyte proliferation by µ opioids: integration of G protein and β-arrestin 2-dependent pathways
Figure Lengend Snippet: Inhibition of µ-opioid or EGF-induced ERK phosphorylation in immortalized astrocytes. (a) Cells were transfected with MOR cDNA, serum-starved, and treated with 1 µM morphine (Morph) or DAMGO, followed by EGF (50 ng/mL, 3 min) and ERK1/2 phosphorylation was assayed. In these and following figures of ERK assays, gels shown are representative immunoblots showing phosphorylated and total ERK1/2. EGF stimulated ERK1/2 [15 ± 1.6 fold over controls ( n = 6)]. ERK1/2 phosphorylation was assayed. EGF stimulation of ERK was 10–15 fold over controls at concentrations of 10, 20, 50, and 100 ng/mL in immortalized astrocytes. * p
Article Snippet: Chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA) with the following exceptions: CTAP, DAMGO, morphine, and norbinaltorphimine were obtained from NIDA Drug Supply (Research Triangle, NC, USA); Protein G plus/Protein A-Agarose beads, EGF, AG1478, and W-7 were obtained from Calbiochem (San Diego, CA, USA); trypsin-EDTA solution was obtained from Gibco (Carlsbad, CA, USA); PTX was obtained from List Biological Laboratories, Inc. (Campbell, CA, USA); Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from ATCC (Manassas, VA, USA); anti-phospho-ERK1/2 (p-ERK; directed against phospho Thr202/Tyr204) antibody (Ab), anti-EGFR Abs for immunoprecipitation and immunoblotting, and antiphosphoTyr Ab were obtained from Cell Signaling Technology (Beverly, MA, USA); anti-glial fibrillary acidic protein (GFAP) Ab was obtained from ImmunoStar, Inc. (Catalog #:22522; Hudson, WI, USA); anti-β-arrestin2 and anti-ERK Ab were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-TuJ 1 Ab was obtained from Neuromics (Edina, MN, USA); FuGENE 6, 5′-bromo-2′-deoxy-uridine (BrdU), and the Abs for its detection from the BrdU Labeling and Detection Kit I was obtained from Roche (Basel, Switzerland), Alexa Fluor-labeled secondary Abs and horse serum were obtained from Invitrogen (Carlsbad, CA, USA) or Molecular Probes (Eugene, OR, USA); and Vectashield Mounting Medium was obtained from Vector Laboratories, Inc. (Burlingame, CA, USA).
Techniques: Inhibition, Transfection, Western Blot