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
L cycle regulation. The LxCxE motif and the tripeptide HDP of the J domain of the polyomavirus tumor proteins make direct contacts with the Rb proteins and Hsc70, respectively; together they effect the release of members of the E2F family of transcription factors from their Rb partners to promote cell cycle progression [reviewed in 55]. There is less certainty about the identity of tAg amino acids
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
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
Rvation will require additional investigation to determine if enhanced PP2A binding is associated with a transformed phenotype. It should be noted that to detect PP2A binding in this study, we employed antibodies that recognized the catalytic subunit of PP2A. PP2A is found abundantly as either a holoenzyme consisting of the AC core plus a regulatory B subunit, or as the AC core alone. High levels
S where phenotyped on MS medium containing 0 and 3 (w/v) Suc and soil (peat/perlite/vermiculite) according to the parameters described previously (Boyes et al., 2001). Seeds were sterilized with chlorine gas and sown on MS medium containing 3 (w/v) Suc followed by 48 h of stratification at 4 . Plants were grown for 2 weeks at 22 with a light intensity of 80 mmol quanta m22 s21 in a 16-h photop