FIG. 7.

The interaction between myc-Trt1 and Polα is compromised in a polαts13 background. The myc-trt1⁺ (polα⁺) strain and the polαts13 myc-trt1⁺ mutant strain were grown at 25°C. Cells were harvested, and protein extracts were prepared. Asterisks indicate three unrelated bands, which were used as loading controls. (A) Coimmunoprecipitation of myc-Trt1 from myc-trt1⁺ (polα⁺) cells. Extracts from the myc-trt1⁺ cells were immunoprecipitated (IP) with anti-c-myc antibody (lane 1) or anti-Polα antibody (lanes 2 and 3) and probed with anti-c-myc antibody. (B) Coimmunoprecipitation of myc-Trt1 from polαts13 myc-trt1⁺ cells. Extracts from polαts13 myc-trt1⁺ cells were immunoprecipitated with anti-c-myc antibody (lane 1) or anti-Polα antibody (lanes 2 and 3) and probed with anti-c-myc antibody. (C) Comparison of the myc-Trt1 and Polα protein levels and their efficiency at coimmunoprecipitation in myc-trt1⁺ (polα⁺) and polαts13 myc-trt1⁺ cells. Cell extracts were diluted to 1, 0.5, 0.25, and 0.1 mg of total protein extract of myc-trt1⁺ (polα⁺) and polαts13 myc-trt1⁺ cells. One percent of each diluted extract was loaded onto SDS gels followed by Western blotting with anti-Polα antibody to determine the level of Polα proteins or anti-myc antibody to determine the level of myc-Trt1 protein. Polα proteins were immunoprecipitated with anti-Polα antibody from the same diluted extracts as in the three top lanes: myc-trt1⁺ (polα⁺) and polαts13 myc-trt1⁺. Ten microliters of anti-Polα immunoprecipitates was loaded onto SDS gel followed by Western blotting with anti-Polα antibody to determine the levels of Polα protein and with anti-myc antibody for coimmunoprecipitation of Polα and myc-Trt1.