Phosphorylation and nuclear accumulation of Dicer upon DNA damage in HEK293 cells. (A) Detection of phosphorylated (autoradiograph, p-Dicer) or total Dicer (immunoblot, A-2) immunoprecipitated with 13D6 from whole cell extracts (WCE) after ³²P-orthophosphate metabolic labeling in the absence or presence of calf intestine phosphatase (CIP). CIP signals, silver stain; Eto., etoposide; H₂O_2, hydrogen peroxide; IgG, immunoglobulin heavy chain. Immunoblot signals were quantified using ImageJ. (B) Immunoblot showing Dicer-TAP migration by Phos-tag SDS-PAGE immunoprecipitated from whole cell extracts (WCE). IgG, immunoglobulin heavy chain; #, unspecific signal; migration units relative to wells. The entire gel is shown. (C) Immunoblots showing total Dicer (A-2) in subcellular fractions. CP/NP, cytoplasmic/nuclear fraction; fractionation marker: Rad21 and H3, nucleoplasm/chromatin (NP); α-tubulin, cytoplasm (CP); Grp75, mitochondria. (D) Immunoblots detecting phosphorylated histone variant H2A.X (γH2A.X, S139), total (A-2) and phosphorylated (p-DCR-1) endogenous Dicer immunoprecipitated from nuclear lysates using the H212 antibody. GFP, control immunoprecipitation (IP; left). Quantitation of p-DCR-1 IP signals as fold-change over total Dicer IP signals (right). *, P < 0.05; error bars, means ± SEM of three biological replicates. (E) Confocal imaging of phosphorylated (p-DCR-1) and total (13D6) Dicer in wild-type or Dicer-depleted (Dicer KD) cells. All quantifications represent number of cells that have the shown phenotype. (F) Confocal imaging as in E (top) and immunoblots (bottom) of phosphorylated (p-DCR-1) Dicer and γH2A.X after time course kinetics with γ-irradiation. Ponc., Ponceau S staining, loading control; Gy, Gray.