Identification of SMARCAL1 interacting proteins by mass spectrometry.

<p>(A) 293T cells were transfected with a Flag-SMARCAL1 expression vector or an empty vector control. Cells were harvested, lysed and Flag antibody conjugated beads were used to immunopurify SMARCAL1. HeLa cells expressing endogenous SMARCAL1 were harvested, lysed, and SMARCAL1-909 antibody or IgG control antibody were used for immunopurification. Proteins were eluted from beads with either the Flag or SMARCAL1-909 peptides and subjected to 2D-LC-tandem mass spectrometry. Where indicated, the cells were treated with 2 mM HU for 16 h prior to harvesting. The number of peptides identified in each purification is reported. (B–D) HeLa nuclear extracts were prepared. Control IgG, SMARCAL1-909 antibody, or WRN antibody immunoprecipitates (IP) as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Where indicated the nuclear extracts were treated with benzonase prior to the immunoprecipitation. In (D) the cells were either mock treated (Unt.) or incubated with 2 mM HU for 16 h prior to harvesting.