Figure 6.
Figure 6. NPM1 interacts with PIDD-CC and PIDD-N after DNA damage. (A) HeLa cells treated with DMSO, actinomycin D (Act-D; 500 nM) or camptothecin (CPT; 200 µM) were harvested 24 h after treatment, lysed, and immunoprecipitated (IP) with NPM1 antibody. Immunoprecipitates were analyzed by Western blot (IB). (B) HeLa cells treated with DMSO, Act-D (500 nM), or CPT (200 µM) were harvested 24 h after treatment, lysed, and immunoprecipitated with monoclonal Anto-1 antibody to PIDD C terminus. Immunoprecipitates were analyzed by Western blot. (C) HeLa cells treated with or without Gö6976 (1 µM) ± IR (10 Gy) were harvested 24 h after IR, lysed, and immunoprecipitated with monoclonal anti-PIDD antibody. Immunoprecipitates were analyzed by Western blot. (D) HeLa cells treated with or without Gö6976 (1 µM) ± IR (10 Gy) were harvested 24 h after IR, lysed, and immunoprecipitated with anti-NPM1 antibody. Immunoprecipitates were analyzed by Western blot. (E) Schematic representation of NPM1 (WT) and NPM1c+ (mutated in AML) protein domain structure. Indicated are amino acids defining the extremities of deletion constructs used in J. OD/ChD, oligomerization/chaperone domain; D/RBD, DNA/RNA binding domain; Aro, aromatic region; NuLS, nucleolar localization signal; NES, nuclear exclusion signal. (F) Schematic representation of PIDD-FL, PIDD autocleavage products. Indicated are amino acids defining the extremities of deletion constructs used in G and autocleavage sites in PIDD. LRR, LRR domain; ZU-5, ZO-1 and UNC5-like; UPA, uncharacterized protein domain in UNC5, PIDD, and Ankyrin family of proteins (designates the putative PIDD oligomerization domain; Janssens and Tinel, 2012). (G) HeLa cells transfected with the indicated Flag-PIDD constructs were harvested 24 h after transfection. Flag immunoprecipitates were analyzed by Western blot. (H) HeLa cells treated with DMSO, Act-D (500 nM), or CPT (200 µM) were harvested 24 h after treatment, lysed, and immunoprecipitated with S-17 antibody to PIDD N terminus. Immunoprecipitates were analyzed by Western blot. ns, nonspecific band. (I) HeLa cells treated with DMSO, Act-D (500 nM), or CPT (200 µM) were harvested 24 h after treatment, lysed, and immunoprecipitated with NPM1 antibody. Immunoprecipitates were analyzed by Western blot. (J) HeLa cells cotransfected with the indicated HA-NPM1 constructs and the noncleavable mutant form of PIDD were harvested 24 h after transfection. HA immunoprecipitates were analyzed by Western blot.

NPM1 interacts with PIDD-CC and PIDD-N after DNA damage. (A) HeLa cells treated with DMSO, actinomycin D (Act-D; 500 nM) or camptothecin (CPT; 200 µM) were harvested 24 h after treatment, lysed, and immunoprecipitated (IP) with NPM1 antibody. Immunoprecipitates were analyzed by Western blot (IB). (B) HeLa cells treated with DMSO, Act-D (500 nM), or CPT (200 µM) were harvested 24 h after treatment, lysed, and immunoprecipitated with monoclonal Anto-1 antibody to PIDD C terminus. Immunoprecipitates were analyzed by Western blot. (C) HeLa cells treated with or without Gö6976 (1 µM) ± IR (10 Gy) were harvested 24 h after IR, lysed, and immunoprecipitated with monoclonal anti-PIDD antibody. Immunoprecipitates were analyzed by Western blot. (D) HeLa cells treated with or without Gö6976 (1 µM) ± IR (10 Gy) were harvested 24 h after IR, lysed, and immunoprecipitated with anti-NPM1 antibody. Immunoprecipitates were analyzed by Western blot. (E) Schematic representation of NPM1 (WT) and NPM1c+ (mutated in AML) protein domain structure. Indicated are amino acids defining the extremities of deletion constructs used in J. OD/ChD, oligomerization/chaperone domain; D/RBD, DNA/RNA binding domain; Aro, aromatic region; NuLS, nucleolar localization signal; NES, nuclear exclusion signal. (F) Schematic representation of PIDD-FL, PIDD autocleavage products. Indicated are amino acids defining the extremities of deletion constructs used in G and autocleavage sites in PIDD. LRR, LRR domain; ZU-5, ZO-1 and UNC5-like; UPA, uncharacterized protein domain in UNC5, PIDD, and Ankyrin family of proteins (designates the putative PIDD oligomerization domain; Janssens and Tinel, 2012). (G) HeLa cells transfected with the indicated Flag-PIDD constructs were harvested 24 h after transfection. Flag immunoprecipitates were analyzed by Western blot. (H) HeLa cells treated with DMSO, Act-D (500 nM), or CPT (200 µM) were harvested 24 h after treatment, lysed, and immunoprecipitated with S-17 antibody to PIDD N terminus. Immunoprecipitates were analyzed by Western blot. ns, nonspecific band. (I) HeLa cells treated with DMSO, Act-D (500 nM), or CPT (200 µM) were harvested 24 h after treatment, lysed, and immunoprecipitated with NPM1 antibody. Immunoprecipitates were analyzed by Western blot. (J) HeLa cells cotransfected with the indicated HA-NPM1 constructs and the noncleavable mutant form of PIDD were harvested 24 h after transfection. HA immunoprecipitates were analyzed by Western blot.

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