Extended Data Fig. 4: Translation stalling does not strongly contribute to the diminished expression of SMAD4 harboring nonstop extension mutations.
a, SMAD4 WT and nonstop extension mutations and a control missense mutant (G1082A) encoding vectors were subjected to coupled in vitro transcription/translation reactions (IVT). The IVT reactions were analyzed by western blotting and probed with anti-SMAD4 antibodies. b, Bands from (a) were quantified and presented as % of WT-SMAD4. c, SMAD4 transcripts from the IVT reactions were quantified by RT-qPCR, normalized to rabbit 18S rRNA and presented as relative expression normalized to WT. d, Schematic representation of the translation stalling in vivo reporters. e, Reporter plasmids containing no stalling sequence in the linker between GFP and mCherry (control), the positive control inducing stalling with 20 lysine codons ((AAA)20), a negative control stem loop or the SMAD4 extension were transfected into BxPC3 cells and analyzed by flow cytometry. Scatter plots show mCherry and GFP expression in transfected cells are shown. f, Median fluorescence intensities of mCherry and GFP in GFP-positive cells were determined and their means are depicted as relative mCherry / GFP ratios normalized to control. The gating strategy for flow cytometry is described in Supplementary Fig. 10. All panels: n=3 biologically independent experiments, mean ± SEM, ANOVA with ***p<0.001, **p<0.01, *p<0.05. Numerical source data and unprocessed blots are provided with the paper.
