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1.
FIG. 3

FIG. 3. From: Pta1, a Component of Yeast CF II, Is Required for Both Cleavage and Poly(A) Addition of mRNA Precursor.

Yth1 is not detected in Cft1 immunoprecipitates of CF II. Cell extract (lanes 2 to 4) or a CF II phosphocellulose fraction (lane 5) was immunoprecipitated with the indicated antibodies, and blots of the immunoprecipitates were probed with antibody against Cft1, Pap1, or Yth1. Recombinant Yth1 produced in E. coli is shown in lane 1, and an immunoprecipitation with preimmune serum is shown in lane 2.

Jing Zhao, et al. Mol Cell Biol. 1999 November;19(11):7733-7740.
2.
FIG. 5

FIG. 5. From: Pta1, a Component of Yeast CF II, Is Required for Both Cleavage and Poly(A) Addition of mRNA Precursor.

Full-length Pta1 protein is found in a Pap1-containing complex. An equal amount of extract from wild-type (WT) or pta1-1 mutant cells was subjected to immunoprecipitation with monoclonal antibody against Pap1. The immunoprecipitated proteins were fractionated on an SDS–8% polyacrylamide gel, transferred to a polyvinylidene difluoride membrane, and immunostained with antibody against Pta1 (A), Cft1/Yhh1 (B), Brr5/Ysh1 (C), or Pap1 (D). Lane 1, wild-type extract; lane 2, pta1-1 mutant extract; lane 3, wild-type extract immunoprecipitated with anti-immunoglobulin G (IgG) antibody.

Jing Zhao, et al. Mol Cell Biol. 1999 November;19(11):7733-7740.
3.
FIG. 8

FIG. 8. From: Pta1, a Component of Yeast CF II, Is Required for Both Cleavage and Poly(A) Addition of mRNA Precursor.

Pta1 is required for both cleavage and poly(A) addition of CYC1 pre-mRNA substrate. Reactions were carried out as described in the legend to Fig. 6. (A) Coupled cleavage-polyadenylation assays in the presence of ATP. (B) Cleavage assays with 2′-dATP. 32P-labeled CYC1 substrates were combined with extracts of wild-type (WT) (lane 2), pta1-1 (lane 3), and pta1-2 (lane 4) strains and pta1-1 and pta1-2 strains plus CF II (lanes 5 and 6, respectively). In lane 6, a reaction was performed with CF II only. See the Fig. 6 legend for symbol definitions.

Jing Zhao, et al. Mol Cell Biol. 1999 November;19(11):7733-7740.
4.
FIG. 4

FIG. 4. From: Pta1, a Component of Yeast CF II, Is Required for Both Cleavage and Poly(A) Addition of mRNA Precursor.

Immunoblot analysis of cell extract from wild-type and pta1 mutant strains. Equal amounts of protein (15 μg) from wild-type (lanes 1 and 2), pta1-1 (lane 3), or pta1-2 (lane 4) extracts were separated on an SDS–8% polyacrylamide gel, transferred to a polyvinylidene difluoride membrane, and immunoblotted with a monoclonal antibody against Pta1 (A), Cft1/Yhh1 (B), Brr5/Ysh1 (C), or Pap1 (D). Lane 1 is a control blot probed with preimmune antiserum from a mouse subsequently immunized with Pap1 antigen.

Jing Zhao, et al. Mol Cell Biol. 1999 November;19(11):7733-7740.
5.
FIG. 2

FIG. 2. From: Pta1, a Component of Yeast CF II, Is Required for Both Cleavage and Poly(A) Addition of mRNA Precursor.

CF II interacts with Pap1 and Fip1. Whole-yeast-cell extract was used for coimmunoprecipitation. Samples were analyzed by electrophoresis on an SDS–8% polyacrylamide gel and stained with silver. (A) Immunoprecipitation assay with antibody against Pap1 (lane 3) or with preimmune serum (lane 2). (B) Immunoprecipitation with antibodies against Fip1 brought down the same set of proteins as shown in panel A (lane 1). Treatment with preimmune serum is shown in lane 2. (C) Immunoprecipitation assay with antibody against Pap1, followed by a more stringent wash step. All CF II subunits were retained along with Fip1 and Pap1 (lane 3). Numbers to the left of panels A and C indicate molecular masses in kilodaltons.

Jing Zhao, et al. Mol Cell Biol. 1999 November;19(11):7733-7740.
6.
FIG. 7

FIG. 7. From: Pta1, a Component of Yeast CF II, Is Required for Both Cleavage and Poly(A) Addition of mRNA Precursor.

Complementation of polyadenylation activity in the pta1-1 mutant extract. Reactions were performed as described in the legend to Fig. 6, and reaction mixtures were incubated at 30°C for 30 min. 32P-labeled GAL7-1 RNA was incubated with extracts of wild-type (WT) (lane 2), pta1-1 (lane 3), brr5-1 (lane 4), rna14-1 (lane 5), and fip1-1 (lane 6) strains. Lanes 7 to 9 are reaction mixtures containing pta1-1 extracts combined with other mutant extracts as indicated. Lanes 10 to 14 are reaction mixtures containing different mutant extracts or buffer as indicated and supplemented with CF II. The reaction mixture of rna14-1 extract in the presence of a CF I fraction (from the heparin-Sepharose step) is shown in lane 15. See the Fig. 6 legend for symbol definitions.

Jing Zhao, et al. Mol Cell Biol. 1999 November;19(11):7733-7740.
7.
FIG. 6

FIG. 6. From: Pta1, a Component of Yeast CF II, Is Required for Both Cleavage and Poly(A) Addition of mRNA Precursor.

Pta1 is required for both cleavage and poly(A) addition of GAL7 pre-mRNA substrate. Equal amounts of protein were mixed with 32P-labeled GAL7 substrate and incubated at 30°C for 20 min as described in Materials and Methods. Products were resolved on denaturing polyacrylamide gels and visualized by autoradiography. (A) Processing assay with full-length GAL7-1 RNA substrate and ATP. (B) Cleavage assay in which ATP is replaced with 2′-dATP to block the poly(A) addition step. (C) Poly(A) addition assay with precleaved RNA substrate GAL7-9. Lanes 1, precursor; lanes 2, wild-type (WT) extract; lanes 3, pta1-1 mutant extract; lanes 4, pta1-2 mutant extract; lanes 5, pta1-1 extract supplemented with CF II; lanes 6, pta1-2 extract supplemented with CF II; lanes 7, reaction with CF II only. The positions of the full-length GAL7-1 precursor RNA, poly(A)+ RNA, upstream and downstream cleavage products, and precleaved GAL7-9 precursor (top to bottom, respectively) are indicated by the bars on the left of each panel.

Jing Zhao, et al. Mol Cell Biol. 1999 November;19(11):7733-7740.
8.
FIG. 1

FIG. 1. From: Pta1, a Component of Yeast CF II, Is Required for Both Cleavage and Poly(A) Addition of mRNA Precursor.

Pta1 is tightly associated with the CF II complex. (A) Fractions containing CF II from Q-Sepharose (QS) and poly(A)-Sepharose (PA) fractionations were subjected to immunoprecipitation with anti-Cft1 antibodies. The immunoprecipitated proteins were resolved on an SDS–8% polyacrylamide gel, and the gel was stained with silver. Four polypeptides, with sizes corresponding to those of CF II subunits, were coprecipitated (lanes 5 and 6). Lanes 3 and 4 are the unbound proteins from the supernatant. Lane 1 contains molecular mass standards (in kilodaltons), and lane 2 is an immunoprecipitation of the Q-Sepharose fraction with preimmune serum. The positions of the light and heavy chains of the immunoglobulins in lanes 2, 5, and 6 are indicated. (B) Immunoblot analysis of the CF II fraction from the Superose 6 step of CF II purification (lane 2) and proteins immunoprecipitated from the Q-Sepharose fraction with anti-Cft1 antibodies (lane 3). Blots were stained with a mixture of Brr5/Ysh1 polyclonal antibodies and a monoclonal antibody against Pta1.

Jing Zhao, et al. Mol Cell Biol. 1999 November;19(11):7733-7740.

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