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68-667 mutants. Pst I sites were engineered at amino acids 4 and 68 with degenerate polymerase chain reaction primers. The Pst I fragment was excised, and the COOH-terminal cGKIa was religated and transformed into yeast strain Y190. The proper reading frame was verified by DNA sequencing. Expression of the COOH-terminal construct in PC97 was confirmed by a protein immunoblot of the Y190 lysate, using the rabbit polyclonal Gal4 DNA-binding domain antibody (Upstate Biotechnology, Lake Placid, NY).
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note
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2, 2 mM EDTA, 2 mg/ml N-dodecyl-B-maltoside, 0.4 mg/ml cholesteryl hemisuccinate, 0.6 M NaCl, 10 mM Na Molybdate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 μg/ml chymostatin, 200 μg/ml aprotinin, 50 μg/ml leupeptin] and incubated for 1 hour at room temperature. Lysates were microfuged for 15 mm at 4°C, and the supernatant was incubated with 100 μI of GST-fusion protein beads overnight, followed by washing in RIPA buffer containing 1% NP40, and boiling for 5 min in SDS sample buffer. Associated proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with either rabbit anti-human cGPK-CT (Upstate Biotechnology, Lake Placid, NY) or rabbit anti-MBS antibody (Berkelely Antibody Company). The membranes were developed with ECL (Amersham Life Science).
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0028358761
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Fluorescence spectroscopy was performed as previously described for the binding of cGK and vimentin (L. A. MacMillan-Crow and T. M. Lincoln, Biochemistry 33, 8035 (1994)].
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(1994)
Biochemistry
, vol.33
, pp. 8035
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MacMillan-Crow, L.A.1
Lincoln, T.M.2
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47
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13044280878
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note
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4,5A were each cotransformed with AL9 in yeast strain Y190, and reporter gene activation assayed as in (9).
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48
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13044304651
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2. 2.5 mM EDTA, 1% Triton X and protease inhibitors as in buffer A). The lysate was incubated for 1 hour at room temperature, microcentrifuged 5 s, and the supernatant precleared with 12-5 μg rabbit IgG followed by protein A beads The precleared supernatant was incubated with either rabbit nonimmune IgG, or rabbit polyclonal anti-MBS (Berkeley Antibody Company) overnight, followed by harvest with protein A beads. Equal amounts of rabbit nonimmune and anti-MBS antibodies were added, and verified by Ponceau staining. SDS-PAGE and cGK immunoblots were performed as above.
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49
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13044274265
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32P-myosin light chains were used as substrate in place of phosphorylase a, as previously described (4), and similar results were obtained Data are presented as mean ± standard error.
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50
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0024517280
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H. Ishihara et al , Biochem. Biophys. Res. Commun. 159, 871 (1989); D. L Brautigan and C. L Shriner, Methods Enzymol. 159, 339 (1988); P. Cohen, Methods Enzymol 201, 389 (1991).
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(1989)
Biochem. Biophys. Res. Commun.
, vol.159
, pp. 871
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Ishihara, H.1
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51
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0023761334
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H. Ishihara et al , Biochem. Biophys. Res. Commun. 159, 871 (1989); D. L Brautigan and C. L Shriner, Methods Enzymol. 159, 339 (1988); P. Cohen, Methods Enzymol 201, 389 (1991).
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(1988)
Methods Enzymol.
, vol.159
, pp. 339
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Brautigan, D.L.1
Shriner, C.L.2
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52
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0025998844
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H. Ishihara et al , Biochem. Biophys. Res. Commun. 159, 871 (1989); D. L Brautigan and C. L Shriner, Methods Enzymol. 159, 339 (1988); P. Cohen, Methods Enzymol 201, 389 (1991).
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(1991)
Methods Enzymol
, vol.201
, pp. 389
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Cohen, P.1
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53
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0017387814
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32P]ATP, for 15 min at 30°C SDS-PAGE was performed as above cGK-CA was prepared by incubating 50 μg of bovine cGKIα [purified as in T. M. Lincoln, W. L Dills, J. D. Corbin, J. Biol. Chem. 252, 4269 (1977)] with 1 μg of trypsin for 3 min at 30°C The reaction was terminated by the addition of 5 μg of soybean trypsin inhibitor. Image analysis of gel bands was performed using Scion Image software, and data are presented as mean ± SE
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(1977)
J. Biol. Chem.
, vol.252
, pp. 4269
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Lincoln, T.M.1
Dills, W.L.2
Corbin, J.D.3
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55
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0019321405
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C. E. Monken and G. N.Gill, J. Biol. Chem. 255, 7067 (1980); W. G. Heil, W Landgraf, F. Hofmann, Eur. J Biochem 168, 117 (1987).
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(1980)
J. Biol. Chem.
, vol.255
, pp. 7067
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Monken, C.E.1
Gill, G.N.2
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0023427390
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C. E. Monken and G. N.Gill, J. Biol. Chem. 255, 7067 (1980); W. G. Heil, W Landgraf, F. Hofmann, Eur. J Biochem 168, 117 (1987).
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(1987)
Eur. J Biochem
, vol.168
, pp. 117
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Heil, W.G.1
Landgraf, W.2
Hofmann, F.3
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13044264559
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Human saphenous vein smooth muscle cells of passage 2-4 were grown on coverslips, fixed with 3% paraformaldehyde then permeabilized with 0.3% Triton X 100. For preservation of stress fiber architecture prior to fixation, cells were washed on ice with PBS for 1 min and permeabilized with 0.3% Triton X in 50 mM tris (pH 7.4) with 0.5 mM PMSF, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 1 μg/ml pepstatin on ice for 1 min The cells were then washed with PBS and fixed as above. For both protocols, cells were blocked with 10% donkey serum in PBS for 1 hour at 37°C, and washed with PBS. Primary antibody mixtures were rabbit polyclonal anti-MBS (1/125), or goat polyclonal anti-cGK (1/250). Secondary antibodies were donkey anti-rabbit IgG-conjugated Cy3 (Amersham Life Science) (1/800) and donkey anti-goat IgG-conjugated fluorescein isothiocyanate (Chemicon International Inc) (1/100). Following incubation with secondary antibody, the coverslips were washed with PBS and mounted in Slow Fade (Molecular Probes).
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0032574821
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G.-R. Wang, Y. Zhu, P V. Halushka, T M. Lincoln, M. E Mendelsohn, Proc. Natl. Acad. Sci. U.S.A. 95, 4888 (1998).
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(1998)
Proc. Natl. Acad. Sci. U.S.A.
, vol.95
, pp. 4888
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Wang, G.-R.1
Zhu, Y.2
Halushka, P.V.3
Lincoln, T.M.4
Mendelsohn, M.E.5
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0023746769
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1-59PCI by electroporation. Cells were arrested in DMEM supplemented with insulin, ascorbic acid and transferrin, then stimulated with 2 μM U46619 for 1 minute with or without 20-min pretreatment with 1 mM 8-Br-cGMP Following treatment, cells were precipitated with TCA, washed with acetone, and protein was subjected to glycerolurea electrophoresis [D. A. Taylor and J. T. Stull. J. Biol. Chem. 263, 14456 (1988)]. The nonphosphorylated and phosphorylated forms of myosin light chain were detected by immunoblotting with monoclonal anti-myosin light chain antibody (Sigma, clone MY-21). Protein bands were analyzed by densitometry Statistical analysis was performed using Student-Newman-Keuls method.
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(1988)
J. Biol. Chem.
, vol.263
, pp. 14456
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Taylor, D.A.1
Stull, J.T.2
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We thank D. L. Brautigan for generosity with reagents and for several helpful discussions, M. Vidal for reagents and advice regarding the two hybrid screens, L. J Moss for help with confocal microscopy, and L. A. MacMillan-Crow for assistance in performing the fluorescence spectroscopy. Supported in part by NIH grants HL09330 (H.K.S.) and HL5S309 (M.E.M.). M.E.M is an Established Investigator of the American Heart Association.
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