Figure 7

Radiation-induced Mcl-1 decline and apoptosis was not affected by silencing of 4EBP1. (A, B) Jurkat cells were irradiated with 10 Gy. (A) 24 h after IR cells were lysed and immunoprecipitation was performed using antibodies against 4EBP1 or eIF4E. As negative controls, isotype-matched antibodies (IgG) were used. Equal input was verified by western blot. Interaction of 4EBP1 with eIF4E was enhanced in irradiated cells. (B) 24 h after irradiation, cells were lysed and and a pull-down assay was made using 7-methyl GTP agarose. 4EBP1 with eIF4E bound to 7-methyl GTP cap. (C-E) Jurkat cells were transfected with 1 μM 4ebp1 siRNA or non-targeting (nt) siRNA by electroporation. 48 h later, cells were irradiated with 10 Gy. (C) 24 h after irradiation, cells were lysed. 4EBP1 and Mcl-1 protein levels were analyzed by densitometry and normalized to the respective levels in non-irradiated cells transfected with non-targeting siRNA. Silencing of 4EBP1 had hardly any effect on Mcl-1 protein levels. Dissipation of the mitochondrial membrane potential (ΔΨm low, D) and DNA fragmentation (sub G1, E) were analyzed by flow cytometry 24 h and 48 h after IR, respectively. (F) Jurkat cells were transfected with 250 nM mcl1 siRNA. Lysates were made 3 h and 6 h after electroporation. Mcl-1 protein levels were analyzed by densitometry and normalized to the Mcl-1 level in lysates made 3 h after transfection with non-targeting siRNA. Down-regulation of Mcl-1 by siRNA resulted in a rapid dephosphorylation of 4EBP1 which was indicated by the shift to faster migrating p17 band. Flow cytometric data show mean values ± S.D. (n = 3), n.s. indicates no significance (p > 0.05).