Fig. 2

Key determinants for LAP2α-RPA binding. A Whole-cell lysates from HeLa cells expressing S protein-FLAG-Streptavidin binding peptide (SFB)-tagged RPA1 deletions were immunoprecipitated and immunoblotted with antibodies against the indicated proteins. The RPA1 domain and deletion mutants annotated with residue numbers are shown. DBD represents DNA-binding domain. B Schematic representation of the evolutionarily conserved residues in the linker region of LAP2α. C Schematic representation of the domain structure of LAP2α and its variants. The domains and truncated or deleted variants are marked with the indicated residue numbers. The LEM (LAP2, emerin, MAN1) domain, LEM-like domain, LAP2α-specific C-terminus, and RB or Lamin A/C binding regions are shown. N, N-terminus from amino acid 1 to 185; M, middle region from 186 to 414; C, C-terminus from 415-695. ΔN50, residues from 1-50 are deleted; Δ76-89, residues from 76 to 89 are deleted; and so forth. D–F Whole-cell lysates from HeLa cells expressing FLAG-GFP-tagged LAP2α truncation mutants were immunoprecipitated and immunoblotted with antibodies against the indicated proteins. G Whole-cell lysates from HeLa cells expressing FLAG-GFP-tagged LAP2α deletion mutants were immunoprecipitated and immunoblotted with antibodies against the indicated proteins. H FLAG-tagged LAP2α variants were transfected into HeLa cells followed by co-immunoprecipitation and immunoblotting analysis. RK>AA (90-109) represents that all arginine and lysine residues from amino acid 90 to 109 of LAP2α are simultaneously replaced by alanine, and so forth. I GST pull-down assays with recombinant GST-DBD-A and His-tagged LAP2α variants. GST-DBD-A and His-tagged LAP2α were purified from bacteria cells and insect cells, respectively. J His pull-down assays with recombinant His-tagged LAP2α variants and RPA. RPA (including RPA1, RPA2, and RPA3) and His-tagged LAP2α variants were purified from bacteria cells and insect cells, respectively. The asterisks indicate the recombinant proteins stained by Coomassie brilliant blue