and 33054N to O
and 33054N to O.P.) and by the National Science Foundation International Program (USA-Mexico; to O.P. RNA from cell suspensions treated with 200 mm mannitol for the times indicated. Ethidium bromide stained gel (bottom) PDK1 shows equivalent RNA loading prior to blotting. Blots are representative of three impartial experiments. The 41-kD Polypeptide Is usually a Glycosylated Form of (3 h at 4C) using a Beckman SW 28 swinging bucket rotor in a Beckman L8-M ultracentrifuge (Beckman, Mexico City). On a discontinuous Suc gradient TP from cell suspension cultures separates at the 0/16% Suc interface while PM is usually collected from your 32%/38% Suc interface (Vera-Estrella et 9-Dihydro-13-acetylbaccatin III al., 1999). TP isolated from root and leaf tissue is collected at the 0%/22% Suc interface (Barkla et al., 1999). The purity of the TP portion was calculated by assaying for the relative contribution of PM, TP, and mitochondrial ATPase enzyme activities. From these studies it is estimated that the 0%/16% Suc interface had 85% bafilomycin and nitrate-sensitive ATPase activity (attributed to 9-Dihydro-13-acetylbaccatin III V-ATPase activity around the TP). No mitochondrial marker activity was detected (data not shown). Bands from your discontinuous gradient or fractions (0.5 mL) from your continuous Suc gradient were collected, frozen in liquid N2, and stored at ?80C. The Suc concentration of fractions from continuous gradients was measured using a Zeiss refractometer (Zeiss, Mexico City). Previously we have shown that ice herb cell suspensions show similar adaptive responses as those of leaves to salt and osmotic stress 9-Dihydro-13-acetylbaccatin III (Vera-Estrella et al., 1999). Pulse-Chase Labeling and Extraction of Total Protein Cells of ice plant labeled for 1 h with 35S Met/Cys followed by incubation from 0.5 to 5 h 9-Dihydro-13-acetylbaccatin III in the presence of 200 mm mannitol, were filtered onto Whatman Number 1 1 filter paper, frozen in liquid N2, homogenized in extraction buffer (100 mm Tris-MES, pH 8.0, 1 mm EGTA, 5 mm dithiothreitol, 4 mm MgSO4, 5% [w/v] insoluble PVP), and vortexed for 1 min. The samples were then filtered through one layer of Miracloth (Calbiochem, La Jolla, CA), and the crude protein extracts were centrifuged at 10,000for 15 min using a SS34 rotor in a Sorvall 5C high speed centrifuge (DuPont, Newton, CT) to remove cellular debris. Samples were utilized for immunoprecipitation (observe below) and resolved by SDS-PAGE on 12.5% (w/v) linear acrylamide gels. Signals were detected with a PhosphorImager (ImageQuant, Molecular Dynamics, Sunnyvale, CA). Preparation of Template DNA, In Vitro Transcription, and Capping of mRNA The coding region of was cloned into the pGEM-HE vector, while for 20 s and the supernatant eliminated using a 1-mL syringe. The protein pellet was washed twice with NET-gel buffer followed by a final rinse with wash buffer (10 mm Tris/HCl, pH 7.5, 0.1% Nonidet P-40). The pellet was then resuspended with 20 prior to loading onto a 12.5%-linear acrylamide gel for SDS-PAGE and subsequent protein blotting. SDS-PAGE and Protein Immunoblotting Samples were prepared according to the method of Parry et al. (1989). Protein was precipitated by dilution of the samples 50-fold in 1:1 (v/v) ethanol:acetone and incubated overnight at ?30C. Samples were then centrifuged at 13,000for 20 min at 4C using an F2402 rotor in a GS-15R table-top centrifuge (Beckman). Pellets 9-Dihydro-13-acetylbaccatin III were air dried, resuspended with Laemmli (1970) sample buffer (2.5% SDS final concentration), and heated at 60C for 2 min before loading onto 12.5%-(w/v) linear acrylamide mini-gels. Unless stated in the physique legends, 12 em /em g of protein was loaded per lane. After electrophoresis, the gels were either stained with Coomassie Amazing Blue R250 (0.25% [w/v] in 50% [v/v] methanol/7% [v/v] acetic-acid), destained in 10% methanol/10% acetic-acid (v/v), and dried under vacuum at 80C for 2 h, or prepared for immunoblotting. SDS-PAGE-separated proteins were electrophoretically transferred onto nitrocellulose membranes (ECL, Amersham, Buckinghamshire, UK) as previously explained (Vera-Estrella et al., 1999). Following transfer, proteins were stained with Ponceau S protein stain (0.1% w/v in 1% v/v acetic acid for 30 s) to check for equal loading/transfer of proteins. Membranes were then blocked with TBS (100 mm Tris, 150 mm NaCl) made up of 0.02% (w/v) Na-azide, and 5% (w/v) fat-free milk powder for 2 h at room temperature. Blocked membranes were incubated for a minimum of 3 h at room temperature with the appropriate primary antibodies, followed by the addition of a 1:5,000 dilution of secondary antibodies (goat anti-rabbit or -mouse) conjugated to horse radish ( em Armoracia lapathifolia /em ) peroxidase. Immunodection was carried out using the chemiluminescent ECL western-blotting analysis system (Amersham, UK)..