Figure 6. PA domain is an elongated... | Rockefeller University Press

Figure 6.

$Figure 6. PA domain is an elongated homodimer. (A) CC1–325-3HA coimmunoprecipitated with CC1–325-13Myc and vice versa. Total extracts from diploid strains expressing GAL1p-induced CC1–325-13Myc or CC1–325-3HA or both were immunoprecipitated (IP) with anti-HA (left) or anti-Myc antibody (right). Bound proteins were probed as indicated. (B) Purification of CC95–303. Lysate denotes bacterial extract expressing CC95–303-S-TEV-Z (indicated by an asterisk), and S-200 indicates pooled fractions of CC95–303-S after TEV digestion and Sephacryl S-200 gel filtration chromatography. (C) Analytical gel filtration of CC95–303-S. Elution of CC95–303-S from a calibrated Sephacryl S-200 column was analyzed by Western blotting with an anti–S tag antibody (top blot) or by Coomassie staining (bottom blot). CC95–303-S migrates as a single peak with an apparent molecular mass of 232 kD and a Stokes radius of 5.07 nm. A.U., arbitrary unit. (D) Sedimentation equilibrium of S-200–purified CC95–303-S at 28,000 rpm. The distribution of CC95–303-S at equilibrium was fitted using the nonlinear least square method of HeteroAnalysis (Cole, 2004). Mass = 58,184 D. Residuals from fitting are shown at the bottom.$

PA domain is an elongated homodimer. (A) CC^1–325-3HA coimmunoprecipitated with CC^1–325-13Myc and vice versa. Total extracts from diploid strains expressing GAL1p-induced CC^1–325-13Myc or CC^1–325-3HA or both were immunoprecipitated (IP) with anti-HA (left) or anti-Myc antibody (right). Bound proteins were probed as indicated. (B) Purification of CC^95–303. Lysate denotes bacterial extract expressing CC^95–303-S-TEV-Z (indicated by an asterisk), and S-200 indicates pooled fractions of CC^95–303-S after TEV digestion and Sephacryl S-200 gel filtration chromatography. (C) Analytical gel filtration of CC^95–303-S. Elution of CC^95–303-S from a calibrated Sephacryl S-200 column was analyzed by Western blotting with an anti–S tag antibody (top blot) or by Coomassie staining (bottom blot). CC^95–303-S migrates as a single peak with an apparent molecular mass of 232 kD and a Stokes radius of 5.07 nm. A.U., arbitrary unit. (D) Sedimentation equilibrium of S-200–purified CC^95–303-S at 28,000 rpm. The distribution of CC^95–303-S at equilibrium was fitted using the nonlinear least square method of HeteroAnalysis (Cole, 2004). Mass = 58,184 D. Residuals from fitting are shown at the bottom.