Fig 2: Radiation-induced TTP degradation and TNF-α secretion is inhibited significantly by the proteasome inhibitor MG132.(A) MH-S cells were metabolically labeled with 35S-methionine, were either sham-irradiated or irradiated with 4 Gy, and chased with cold methionine for indicated time periods. After the chase period cell lysates were subjected to immunoprecipitation using TTP antibody, immunocomplexes were resolved by SDS-PAGE and autoradiography. (B) TTP protein’s half life in sham-irradiated (-RT) and irradiated (4 Gy) groups were determined by densitometric scanning of the autoradiographs followed by quantitation using Image J1.32j software (NIH, Bethesda, MD). Relative protein levels were determined in comparison to sample isolated immediately after the pulse labeling (0 h chase). (C) MH-S cells were either sham-irradiated or radiated with 4 Gy, and 44 h after radiation 2 µM of MG132 was added. Cell lysates were collected 4 h after MG132 addition and immunoblotted for TTP and TNF-α. GAPDH was used as loading control.

Fig 3: Radiation caused increased phosphorylation and down-regulation of TTP in MH-S cells.(A) MH-S cells were either sham irradiated or treated with 4 Gy. Cells were harvested at indicated time points and immunoblotted using indicated antibodies. (B) CHO cells overexpressing mouse TTP were treated with 4 Gy, harvested at different time points, and immunoblotted using indicated antibodies. (C) U2OS cells overexpressing human TTP were irradiated with either 4 or 8 Gy and immunoblotted using indicated antibodies.

Fig 4: Radiation causes significant down-regulation of TTP in irradiated mouse lung.(A) C57BL/6 mice were either sham-irradiated or the whole lung was irradiated with a single dose of 15 Gy. Tissue lysates were prepared from three individual animals at each indicated time points post-radiation and immunoblotted using indicated antibodies. (B) Mouse lung cryo-sections from sham-radiated or 15 Gy single fraction radiated mice were isolated at indicated time points and immune-staining were performed as described in materials and methods.

Fig 5: p38 kinase controls radiation-induced TTP phosphorylation and TNF-α secretion by MH-S cells.(A) CHO cells overexpressing TTP were treated with 4 Gy in the presence of either DMSO (vehicle control) or p38 inhibitor (SB203580), or PI3K inhibitor (Wortmannin), or GSK3ß inhibitors (SB415286, SB216763). Cell lysates were prepared 10 min post-radiation and immunoblotted using indicated antibodies. (B) MH-S cells were pretreated with either p38 or GSK3ß inhibitors as above and cell lysates were prepared 10 min post-radiation and immunoblotted with the indicated antibodies. (C) MH-S cells were irradiated with 4 Gy in the presence or absence of a p38 inhibitor (SB203580), and radiation-induced TNF-α secretion was quantified using ELISA. (D) MH-S cells were treated with either control (C) or TTP (T) siRNA. 24 h post-transfection, cells were either left un-irradiated or radiated with 4 Gy. Culture supernatants were collected 48 h post-radiation, and TNF-α levels were quantified. In the inset, the effectiveness of p38 siRNA is shown in cell lysates isolated from C or T siRNA treated cells.