Figure 4. The catalytic residues of SOX are required for mRNA turnover and generation of the degradation intermediate.

(A–B) Wild-type (WT) SOX or SOX putative catalytic (A) or DNA binding (B) residue mutants were transfected into 293T cells with a GFP reporter. RNA was Northern blotted using a 5′ GFP or 18S probe, and protein lysates were Western blotted for SOX and actin (as a loading control). Graphs depicting mean relative GFP levels ± s.e.m. from >3 experiments in cells transfected with the various mutants are also shown (right). **p<0.01, ***p<0.001, One-way ANOVA followed by Dunnett's test versus (−). (C) 293T cells were transfected with control or Xrn1 siRNAs, followed by a DsRed2 reporter +/− WT SOX or the indicated SOX mutant. Northern blots were performed using a probe directed against the 3′ UTR of the DsRed2 mRNA or 18S (upper panels), and Western blots show expression of the SOX mutants and the level of Xrn1 depletion (lower panels). Arrowhead denotes degradation intermediates.