Specificity of ATM/Chk2 signaling on HuR-mRNA association. (A) Left: ATM wild-type (GM02184^+/+) lymphocytes were pretreated for 2 hours with Chk2 or ATM inhibitors and then exposed to 1 Gy of IR and harvested after 6 hours of further incubation. pChk2 and Chk2 levels were assessed by Western blotting. Middle: Chk2 levels by 72 hours after transfection of HEK-293 cells with either control (Ctrl) or Chk2-directed siRNAs. Right: ATM levels in GM5849 AT fibroblasts stably reintroduced with ATM gene. (B) The association of HuR-mRNA complexes after IR (1 Gy, 6 hours) of cells pretreated with Chk2 or ATM inhibitor (10μM) (left), Chk2 knockdown (middle), and ATM reintroduction (right) was tested by mRNP-IP followed by quantitative RT-PCR. Each reaction was carried out in triplicate and was repeated 3 times. The individual enrichment of each mRNA in HuR IPs was normalized to IgG IPs; afterward, the differences in HuR binding were calculated. GAPDH mRNA background amplification in the IP material served as the internal control. Data are the mean ± SEM from 3 independent experiments.