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. 2007 Mar 19;27(11):3951–3961. doi: 10.1128/MCB.02180-06

FIG. 5.

FIG. 5.

Rph1 is not stably associated with other proteins in yeast extracts. (A) Endogenous Rph1 was Flag tagged, and the wild-type (WT) and Flag-tagged strains were analyzed by Western blotting using Flag-specific antibodies. A signal corresponding to Flag-Rph1 was evident only in the tagged strain. The asterisk indicates a cross-reactive band observed in budding yeast extract. (B) Flag-RHP1 yeast extract was fractionated by size exclusion chromatography, and the Rph1-containing fractions were identified by Flag-specific Western blotting. The * indicates a cross-reacting band found in yeast extracts. Size exclusion chromatography molecular mass markers are indicated above the panel. Rph1 eluted from the size exclusion column with an apparent molecular mass of greater than 440 kDa. (C) Recombinant Rph1 was fractionated by size exclusion chromatography, and the Rph1-containing fractions were identified by Coomassie staining. Size exclusion chromatography molecular mass markers are indicated above the panel, and the calculated radius of Rph1 is given above in nm. rRph1 eluted from the size exclusion column at the same position as endogenous Rph1. (D) Recombinant Rph1 was fractionated over a 5 to 20% sucrose gradient, and Rph1-containing fractions were identified by Coomassie staining. Molecular mass standards are indicted above the panel, and the calculated sedimentation coefficient in S is given above.