Figure 1.

Phosphorylation state of endogenous XRCC1. (a) 2D-PAGE western analysis of immunoprecipitated endogenous XRCC1, mock treated (control) and calf-intestine phosphatase (CIP) treated immunoprecipitates. (b) 2D-PAGE western analysis of whole cell lysates from HeLa cells grown in parallel with and without addition of H₂O₂ (62.5 µM). (c) 2D-PAGE western analysis of whole cell lysates from HeLa cells treated with either 0.25% DMSO (mock), 10 µM 4-AN (PARP inhibitor), 25 µM LY294002 (PI3K inhibitor), 25 µM TBB (CK2 inhibitor), 25 µM SB203580 (p38 MAPK inhibitor), or 25 µM BIRB0796 (p38 MAPK inhibitor). Recombinant IRF3 (Interferon regulatory factor 3) was added to all the lysates to serve as an internal positional reference during picture alignment (dotted lines). IRF3 is depicted only in the 2D-PAGE western analysis of BIRB0796 treated cells for simplicity. Cropped images of one out of two gels with similar patterns is depicted. (d) Quantification of %Head and %Tail (defined in C) using the Kodak Molecular Imaging software version 4.0.1. Ratios of “head” and “tail” densities versus total XRCC1 intensity (head + tail) were calculated after subtracting background intensity values.