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
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
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
Introduced mutations into the conserved CxxxPxC sequence and the unique LxCxE motif within the C-terminus of JCV tAg. Using a coimmunoprecipitation approach, we confirmed our prediction that the P99A mutant tAg would exhibit a loss of PP2A binding activity. This proline is conserved in the tAgs of primate polyomaviruses as well as of mouse polyoma virus.JCV Small t Protein FunctionsFigure 7. JCV t
Introduced mutations into the conserved CxxxPxC sequence and the unique LxCxE motif within the C-terminus of JCV tAg. Using a coimmunoprecipitation approach, we confirmed our prediction that the P99A mutant tAg would exhibit a loss of PP2A binding activity. This proline is conserved in the tAgs of primate polyomaviruses as well as of mouse polyoma virus.JCV Small t Protein FunctionsFigure 7. JCV t
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
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