In vitro NHEJ assay. (A) Determination of NHEJ activities in extracts prepared from 293T, 293T/eIF3f and 293T/eIF3f-hMSH4 cells. DNA end joining reactions were performed by incubation of cell extracts with Sal I-digested plasmid DNA, and these reactions were terminated at the indicated time points. End joining products were separated by agarose gel electrophoresis. ‘S’ signifies linear DNA substrate, ‘D’ for joint dimer, and ‘M’ indicates all other higher order joint products. (B) Analysis of NHEJ activities in 293T extracts complemented with either 5 μg of BSA or 293T/eIF3f-hMSH4 extracts under identical buffer conditions. A representative gel image was shown on the left, and the relative NHEJ activities were quantified and graphed as a function of time (on the right).