FIGURE 10:

TIS11b S334D mutant exhibits enhanced interaction with the decapping complex activator Dcp1a. (A) Sequence alignment of the distal C-terminus of TTP family members, showing that the TTP-CCR4-NOT interaction motif is highly conserved between TTP family members (Fabian et al., 2013). (B) Western blots showing association of TIS11b proteins with endogenous mRNA decay enzymes. Transiently expressed WT or TIS11b mutants were immunoprecipitated from HEK 293T cell extracts, and precipitates were probed for the presence of TIS11b and mRNA decay proteins. The mRNA-stabilizing protein HuR served as negative control and was not associated with TIS11b. NRS: immunoprecipitation reaction using normal rabbit serum in place of TIS11b antibodies. Input: 5% total-cell extract. (C, D) Quantification of Cnot1/TIS11b and Dcp1a/TIS11b ratio in three independent immunoprecipitation experiments. *, Significantly different from WT TIS11b with p < 0.05; **, significantly different from S334A mutant with p < 0.01 (one-way ANOVA).