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FIGURE 4. xStn1 is required for priming on ssDNA template but not essential for bulk chromosomal replication. A, xStn1-immunodepleted or mock-immunodepleted extracts (HSS and NPE) were analyzed by immunoblotting against xStn1. The volume of the loaded extracts is 0.2 μl/lane. Positions of xStn1 protein are indicated. Although the anti-xStn1 antibody detected a single band in immunoblotting experiments using the cell lysates of Sf9 expressing recombinant xStn1, it produced many nonspecific bands in the immunoblotting using the egg extracts. This is probably because the concentration of endogenous xStn1 is so low (5 nm at most, see supplemental Fig. S8) that cross-reactions with proteins other than xStn1 are dominantly detected over the specific interaction between the antibody and xStn1. Unlike xStn1, most of the nonspecific bands detected in the immunoblotting experiments with anti-xStn1 antibody were not immunoprecipitated by anti-xStn1 antibody, suggesting that the antibody immunodepletes proteins with high specificity compared with the immunoblotting experiments (supplemental Fig. S3). Moreover, similar results were obtained when we immunodepleted xStn1 from HSS with two independently immunized anti-xStn1 antibodies that recognized different sets of nonspecific bands (KU003 and KU004, see supplemental Fig. S3), supporting the notion that the biological effects of the immunodepletion observed in B are caused by the depletion of xStn1 or its binding proteins. B, upper, DNA replication assay using pBluescript dsDNA as template. pBluescript was incubated in xStn1-immunodepleted or mock-immunodepleted HSS for 30 min. Then, 2 volumes of xStn1-immunodepleted or mock-immunodepleted NPE containing [α-32P]dCTP was added, and the reaction was incubated further. Aliquots were withdrawn at the indicated time points, and DNA was isolated and subjected to agarose gel electrophoresis. Nucleotide incorporation was visualized by autoradiography. Lower, DNA replication assay using M13 ssDNA as template. M13 was incubated in xStn1-immunodepleted or mock-immunodepleted extracts containing [α-32P]dCTP for the indicated times. Nucleotide incorporation was analyzed as described above. Image published in: Nakaoka H et al. (2012) © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license Larger Image Printer Friendly View

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