Phosphinothricin

Protoplast isolation and transient assays were carried out as described previously (8 (link)). When required, bortezomib [Selleckchem; prepared as a 10 mM stock solution in dimethyl sulfoxide (DMSO)] was added to the protoplast culture medium at 15 hours following transfection to a final concentration of 5 μM; subsequently, the culture was incubated for a further 2 to 3 hours before analysis. When using protoplasts isolated from the CDC48-WT and CDC48-DN transgenic lines, 10 μM estradiol (prepared as a 10 mM stock solution in ethanol) was included in the culture medium throughout the incubation of protoplasts. For YFP fluorescence and immunoprecipitation assays, 0.1 (10⁵ cells) or 1 ml (10⁶ cells) aliquots of protoplasts were transfected with 5 or 100 μg of DNA, respectively; the fluorescence signals were analyzed after 15 to 18 hours.
Transgenic lines carrying the CDC48-DNa, CDC48-WTa, and 6Myc-Ub constructs were generated by Agrobacterium-mediated transformation (49 (link), 50 (link)). Transformants were selected using MS medium containing hygromycin B (50 μg/ml; Melford) or phosphinothricin (10 μg/ml; Duchefa). Approximately 10 independent T₂ lines were analyzed per construct, and one representative line with a single T-DNA insertion (which showed a 3:1 segregation on selective MS medium in the T₂ generation) was chosen for further analysis.

Murashige and Skoog (MS) medium, spectinomycin, and sucrose were purchased from MB cell (CA, U.S.A). Tetracycline, cefotaxime and phosphinothricin were purchased from Duchefa (Haarlem, Netherlands). The pMJ103 vector was constructed by GreenGene BioTech (Yongin, Korea) and the plasmids (MYO51, MYO53) containing LTB and/or CTB were kindly donated from Dr. M.S. Yang (Chonbuk National University). T4 DNA ligase and restriction endonucleases were obtained from Roche (Basel, Switzerland). Gateway BP and LR recombinase systems were purchased from Invitrogen (Breda, Netherlands). A mini trans-blot cell and Mini-Protean III cell were purchased from Bio-Rad (Hercules, CA, USA). Reagents for SDS-polyacrylamide electrophoresis, such as acrylamide, bis-acrylamide, ammonium persulfate, TEMED, prestained molecular weight markers and Coomassie Brilliant Blue R-250 solution were obtained from Bio-Rad (Hercules, CA, USA). Anti-LTB polyclonal antibody was obtained from Abcam (Cambridge, MA, USA). G_M1-ganglioside and anti-rabbit IgG-conjugated alkaline phosphatase were purchased from Santa-Cruz Biotechnology (CA, USA). Thiamine-HCl, myo-inositol, anti-CTB polyclonal antibody, and other reagents such as salts and buffer components were of analytical grade and purchased from Sigma (St. Louis, MO, USA).