Analysis of selected HuR targets after IR exposure. (A-B) ATM wild-type (GM02184^+/+) and ATM null (GM03332^−/−) lymphocytes were either untreated or exposed to 1 Gy of IR and collected 6 hours later (unless otherwise indicated). The selected mRNAs immunoprecipitated using an anti-HuR antibody (demonstrating changes in association with HuR after IR by microarray analysis) were validated by quantitative RT-PCR; the individual enrichment of each mRNA in HuR IPs was normalized to IgG IPs; afterward, the differences in HuR binding were calculated. Background binding of an abundant transcript encoding a housekeeping protein (GAPDH mRNA) served as a loading control. (C) Western blot analysis of the proteins encoded by the HuR target mRNAs shown in panel B, assayed in whole-cell lysates prepared from lymphocytes that were treated with 1 Gy of IR. β-Actin and GAPDH were assessed as loading markers; relative protein abundance was calculated by densitometry and is expressed as percentage change relative to the levels in nonirradiated ATM wild-type cells. (D) HEK-293 cells were transfected with the indicated siRNAs; cells were either untreated or, 24 hours after transfection, IR treated (1 Gy) and collected 6 hours later (p21, MEK2, ZFP36L1, DUSP10) or 18 hours later (FOXO3, MEK1) for Western blot analysis. (E) HuR binding of target messages in multiple wild-type and ATM null lymphocytes treated with IR (1 Gy, 6 hours); wild-type and ATM null cell lines were purchased from Coriell Cell Repositories. Each cell line was acquired from a different donor. (F) The levels of total mRNA genes validated in panel B were measured by quantitative RT-PCR in ATM wild-type IR-treated compared with untreated lymphocytes. All graphs represent the mean plus or minus SEM from 3 independent experiments.