On kit (Promega); 10 mL of radiolabeled protein was added to the reaction, and samples were resolved by SDS-PAGE, dried, and exposed to a phosphor-imaging screen.Plant Material and Growth ConditionsArabidopsis (Arabidopsis thaliana) T-DNA insertional knockout lines for Tric1 (SALK_031707, SALK_112126, and At3g49560) and Tric2 (SALK_136525, SALK_149871, and At5g24650) were obtained from ABRC and sc
Phosphatase activity of PP2A in studies involving a second known binding partner of tAg, the JCV agnoprotein [40]. We were surprised to identify two LxCxE motifs that had been overlooked in the unique region of the JCV tAg; one of these sites is also found in BKV but neither site resides in the corresponding SV40, WUV, KIV or MCV polyomavirus proteins. As predicted, JCV tAg binds members of the re
Phosphatase activity of PP2A in studies involving a second known binding partner of tAg, the JCV agnoprotein [40]. We were surprised to identify two LxCxE motifs that had been overlooked in the unique region of the JCV tAg; one of these sites is also found in BKV but neither site resides in the corresponding SV40, WUV, KIV or MCV polyomavirus proteins. As predicted, JCV tAg binds members of the re
Flanked by Gateway recombination cassettes (Supplemental Table S4) and cloned intoMurcha et al.pDONR201. LR reactions were carried out with C-terminal GFP fusion vectors for GFP localization (Carrie et al., 2009), pDEST14 for in vitro transcription and translation, and pB2GW7 (35S CaMV promoter) for Agrobacterium tumefaciensmediated complementation (Karimi et al., 2002). Yeast two-hybrid vectors w
Mitochondrial pellet was resuspended in digitonin buffer (30 mM HEPES-KOH, pH 7.4, 150 mM potassium acetate, 10 [v/v] glycerol, and 1 mg of digitonin) and kept on ice for 30 min. Ten microliters of antibody serum, 50 mL of Protein A-Sepharose, 1 (w/v) BSA, and Complete protease inhibitor (Roche) were added to the mitochondria and incubated for 6 h at 4 on a rotating wheel at 20 rpm. Beads were