Figure 4. Radiation-induced Ser¹⁸⁶ phosphorylation of TTP primes it for polyubiquitination.

(A) U2OS cells overexpressing either wild-type (WT) or various serine to alanine mutants [S34A, S34/35/39A (3SA) and S186A] of human TTP, were either sham irradiated or treated with 4 Gy. Twenty-four hours post-irradiation cell lysates were immunoblotted. Changes in total TTP levels were calculated assuming TTP overexpressed untreated control band intensity as 1. (B) U2OS cells overexpressing either WT or various (3SA and S186A) mutants of TTP were either sham-irradiated or treated with 4 Gy. Six hours after irradiation cells were treated with MG132 as above, and 4 h post-MG132 treatment cell lysates were immunoprecipitated using TTP antibody and immunoblotted. (C) Representative MS/MS spectrum identifying serine¹⁸⁶ phosphorylation on TTP upon 4 Gy.