Journal:
Article Title: The importance of ERK activity in the regulation of cyclin D1 levels and DNA synthesis in human cultured airway smooth muscle
doi: 10.1038/sj.bjp.0703454
Figure Lengend Snippet: Cyclin D1 mRNA levels measured by Northern blot analysis in response to 16 h treatment with thrombin and bFGF in the absence or presence of PD 98059 (30 μM). Monomed A (1%) was added to all cells. The mRNA from cell extracts was separated on a formaldehyde denaturing gel and transferred to a nylon membrane. Cyclin D1 mRNA was hybridized with a radiolabelled DNA probe and then detected by apposition to X-ray film or phosphorimaging. (a) Representative blot of the responses to the high mitogen concentrations and (b) pooled data, expressed as the mean and s.e.m. of six experiments of identical design from at least three different cell lines. Results are expressed as a fold increment over the mRNA levels in control cells. GAPDH mRNA levels are shown as a control for loading differences (a). *P<0.05, **P<0.01 Responses are compared to the cyclin D1 mRNA levels in control cells stimulated with Monomed A (1%) alone. Repeated measures ANOVA, followed by Dunnet's post-hoc test was used for multiple comparisons (b).
Article Snippet: The compounds used and their sources were as follows: essentially fatty acid-free bovine serum albumin fraction V (BSA), L -glutamine, thrombin (bovine plasma), anti-smooth muscle myosin (mouse monoclonal), HEPES buffer, leupeptin, dithiothreitol, propidium iodide, Tris, sodium deoxycholate, phenylmethylsulfonylfluoride (PMSF), orthovanadate, β-mercaptoethanol (Sigma, U.S.A.); amphotericin B (fungizone), human recombinant basic fibroblast growth factor (Promega, U.S.A.); collagenase type CLS 1, elastase (Worthington Biochemical, U.S.A.); Dulbecco's Modified Eagle's Medium (Flow Laboratories, U.K.); Dulbecco ‘A' phosphate buffered saline (Oxoid, U.K.); foetal calf serum, monomed A, penicillin-G, versene, streptomycin, trypsin (CSL, Australia); Hybond™-C supernitrocellulose membranes, α- 32 P-dCTP (3 mCi mmol −1 ), [6- 3 H]-thymidine (5 mCi mmol −1 ), [γ 32 P]-ATP (3 mCi mmol −1 ), enhanced chemiluminescence reagents, Biotrak™ ERK (p42/p44 MAP kinase) enzyme assay kit, Hyperfilm MP, (Amersham, Cardiff, U.K.); anti-smooth muscle α-actin antibody (mouse monoclonal (M851)) (Dako Corporation, U.S.A.); sheep anti-rabbit IgG horseradish peroxidase-conjugated antibody (Silenus Laboratories, Melbourne, Australia); PD 98059, phospho-specific p42/p44 ERK antibody (rabbit polyclonal) (New England Biolabs, U.K); sodium hydroxide, (Biolab Scientific, Australia); RNaseII (Boehringer, Australia); dimethylsulphoxide (DMSO), EDTA, acetone, sodium dodecyl sulphate (SDS), glycine, methanol, Tween-20 (BDH, U.K.); endothelin-1 (Auspep, Australia); Aprotinin, (Bayer, Germany); X-omat AR film (Kodak, Australia); Triton X-100, sodium chloride, magnesium chloride, glycerol (Ajax, Australia); anti-cyclin D1 antibody (rabbit polyclonal IgG) (Upstate Biotechnology, NY, U.S.A.); HRP-conjugated anti-rabbit IgG secondary antibody (Silenus, Australia); P81 filter paper (Whatmann Int., U.K.); anti-ERK antibody (goat polyclonal IgG, C-16) (Santa Cruz, U.S.A.); 4-(2-aminoethyl)-benzene sulphonyl fluoride hydrochloride (Pefa Bloc) (Boehringer Mannheim, Germany); protein-G Sepharose (Pharmacia Biotech, Sweden); Trizol™ reagant, myelin basic protein (Gibco BRL, Australia); Dynabeads, oligo (dT) 25 (Dynal, Oslo, Norway); Immobilon-Ny + nylon membranes (Millipore, U.S.A).
Techniques: Northern Blot
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