FIGURE 3.

GPS2 proteasomal degradation depends on polyubiquitination at the carboxyl terminus domain. A and B, Western blot (WB) analysis for HA-tagged ubiquitin (Ub) or endogenous ubiquitin showing polyubiquitination of GPS2 as immunoprecipitated (IP) from 293T cells either transfected with HA-Ub (A) or not transfected (B). C, Western blot analysis of full-length GPS2 (aa 1–327) and different deletants expressed in reticulocyte lysate by in vitro transcription and translation (TnT). D, the effect of site-directed mutagenesis of lysines 254, 300, and 327 on GPS2 expression levels in NEs and CEs was measured by Western blotting for HA. Blotting for tubulin and HDAC2 provides controls for CEs and Nes, respectively. E, gene expression analysis in 293T cells comparing the efficiency of GPS2 WT and the more stable triple lysine mutant in rescuing the effect of down-regulating endogenous GPS2 by siRNA on the activation of the proinflammatory target CCL2. Data are presented as mean ± S.D. *, p < 0.08.