Fig 4. LdCdc45 interacts with PCNA via its PIP box.
a and c. Western blot analysis of PCNA immunoprecipitates from lysates of cells expressing a. Cdc45-FLAG ectopically (indicated as Cdc45-FLAG in the figure) c. Cdc45-FLAG or Cdc45-PIP-FLAG ectopically (indicated as Cdc45-FLAG or Cdc45-PIP-FLAG in the figure). Ld1S+vector was used as control (Ld1S cells harboring pXG-FLAG vector which carried the neor drug cassette). IP: immunoprecipitation. IB: immunoblotting (western blot). Immunoblots were probed with anti-FLAG (Sigma, 1:1000 dil), anti-PCNA (raised earlier in the lab, 1:2500 dil) and anti-IgG (Jackson ImmunoResearch, 1:10000 dil) antibodies. Both experiments were carried out twice with similar results; one data set of each is shown. b. and d. Analysis of pull down reactions using b. PCNA and MBP-Cdc45481-785 d. PCNA and MBP-Cdc45481-785 or PCNA and MBP-Cdc45-PIP481-785, by Coomassie staining and western blotting. IB: Immunoblotting. Immunoblots were probed with anti-MBP (Sigma, 1:12000 dil) and anti-PCNA (1:2500 dil) antibodies. Both experiments were done twice with comparable results; one data set of each is shown. e. Analysis of purified MBP-Cdc45481-785 proteins by Coomassie staining of SDS-PAGE (left panel) and western blotting using anti-MBP antibodies (right panel). f. Analysis of pull down reactions using MBP and PCNA, by Coomassie staining and western blotting. g. Analysis of pull down reactions using MBP-Cdc45481-785 proteins (wild type and PIP mutant) and PCNA, by Coomassie staining and western blotting. Both experiments were performed twice with comparable results; one data set of each is shown.