Fig. 1

RPA is physically associated with LAP2α. A SILAC-based quantitative mass spectrometry analysis of RPA1-containing protein complexes with HeLa cells that allow doxycycline (Dox)-inducible expression of stably integrated FLAG-RPA1. Control cells were labelled with “heavy isotopic lysine and arginine” (K6R10) and the cells under Dox (1 ng/μl) treatment were labelled with “light isotopic lysine and arginine” (K0R0). DNase (300 U/ml)-treated cellular extracts were immunopurified with anti-FLAG affinity beads and eluted with FLAG peptide. The eluates were desalted by gel separation and mixed for digestion followed by mass spectrometry analysis. Biological duplicate experiments were performed. B Volcano plot showing the relative enrichment of RPA1-containing protein complex from SILAC-based quantitative mass spectrometry. Each point represents the potential interactor, and the 11 top candidates (fold change > 15 and P value < 0.01) are indicated. C Co-immunoprecipitation analysis of the interaction between LAP2α and RPA. Whole-cell lysates from HeLa cells were pre-treated with DNase followed by immunoprecipitation and immunoblotting with antibodies against the indicated proteins. α, anti-. D Co-immunoprecipitation analysis of the interaction between LAP2α and RPA. Whole-cell lysates from MCF-7 and U2OS cells were pre-treated with DNase followed by immunoprecipitation and immunoblotting with antibodies against the indicated proteins. E Analysis of the binding affinity of recombinant RPA purified from bacteria cells and His-tagged LAP2α purified from insect cells by biolayer interferometry (BLI) assay. The black line indicates fitted curves and the color traces represent raw data. Data are representative of two independent experiments. RPA1, RPA2, and RPA3 of the RPA complex were co-purified and examined by Coomassie brilliant blue staining. F Co-immunoprecipitation analysis of the association of individual recombinant RPA subunit with LAP2α. All proteins were individually purified from insect cells. G Co-immunoprecipitation analysis of the association of recombinant RPA1 with LAP2α in buffer with an increasing amount of ionic strength