Fig 1: ATR activity is hampered upon inhibition of Wee1 in the presence of gemcitabineA, B. Panc1 and U2OS cells were treated with 1μM Wee1i or DMSO in the presence or absence of 300nM gemcitabine for 24 h. Cells were then fixed and stained for phosphorylated Rad17 (another ATR-substrate). Fluorescence intensities were determined by automated microscopy (BD Pathway). Error bars represent the SD, n = 3. Images of phospho-Rad17 staining are shown in (Supplemental Figure S4C, D). C, D. Panc1 and U2OS cells were treated with 1μM Wee1i or DMSO in the presence or absence of 300nM gemcitabine for 24 h. Cells were harvested and immunoprecipitation (IP) of ATR was performed. Phosphorylated ATR (Thr1989) was stained on immunoblots (IB), in the cell lysates (Input, C), and after ATR IP D. The Immunoglobulin G heavy chain (IgG-H) of the precipitating antibody was detected by the secondary IB antibody and shown as a loading control.