FIG. 4.

Gadd153 down-regulates Bcl2. (A) Western blot demonstrating that Bcl2 protein levels are diminished in cells constitutively overexpressing Gadd153, while Grp78 expression is unaffected by constitutively elevated Gadd153 levels. The RT-PCR shows that the bcl2 mRNA level is also decreased basally in A94 cells. The gapdh RT-PCR was performed as a control for RNA integrity and loading. (B) Western blot demonstrating down-regulation of Bcl2 in gadd153^−/− Gadd153i cells but not in gadd153^−/− Neo cells. Concomitant with induction of Gadd153 expression by inclusion of 5 mM IPTG in the culture medium, Bcl2 expression decreases, while expression of Grp78 remains constant. In contrast, IPTG had no effect on Bcl2 protein levels in the vector control cells. There was no Gadd153 expression in the gadd153^−/− Neo cells, so no blot is shown for this protein. (C) RT-PCR demonstrating that inclusion of IPTG in the culture medium of gadd153^−/− Neo cells has no effect on bcl2 mRNA levels. In contrast, IPTG-mediated induction of Gadd153 in gadd153^−/−, Gadd153i cells results in diminished abundance of bcl2 mRNA. (D) Northern blot analysis showing that 5 mM IPTG pretreatment for 18 h, which induces Gadd153 expression in gadd153^−/− Gadd153i cells, does not influence the induction of grp78 RNA levels in response to ER stress incurred by 1 μM thapsigargin (TG) for 6 h.