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2-terminal third of NPH1 including the LOV1 domain, designated NPH1N, to the Eco RI-Pst I site of the vector pMal-c2 (New England Biolabs) as a translational fusion to the maltose binding protein (MBP). MBP-NPH1N was expressed in Escherichia coli, purified by amylose-affinity chromatography, and used to prepare polyclonal antibodies in rabbits (Cocalico Biologicals, Reamstown, PA). Immunoblots were analyzed with anti-NPH1 (1:1000 dilution) using the color development method (Promega) with antibody to rabbit immunoglobulin G conjugated to alkaline phosphatase as the secondary antibody.
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Spectroscopic analysis was performed with a Beckman DU-70 spectrophotometer and a Photon Technology International Alphascan spectrofluorometer. Fluorescence excitation spectra were obtained by monitoring the emission at 535 nm. Fluorescence emission spectra were measured by using an excitation wavelength of 390 nm. For fluorescence measurements, insoluble fractions (1 mg) were treated with 6 M guanidine-HCl, 0.1 M sodium phosphate, and 0.01 M tris-HCl (pH 8.0). For absorption studies, insoluble fractions were treated with 10% trichloroacetic acid. In each case, samples were centrifuged at 16,000g for 10 min and the supernatants were analyzed for the presence of chromophore.
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We thank T. Baskin for providing the phototropism action spectrum data, and M. A. Olney and E. Huala for their assistance and critical reading of the manuscript. P.R. thanks the Fondation du 450ue Anniversaire of the University of Lausanne for providing a travel grant. Supported by NSF grants IBM-9219256 and IBM-960114. This paper is Carnegie Institution of Washington, Department of Plant Biology publication 1385.
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