Fig. 2: TRIP12 inhibition does not affect SMAD4 protein stability but reduces its monoubiquitination.

A Western blot comparison of SMAD4 protein levels in indicated TRIP12^+/+ and TRIP12^-/- cell lines. B Western blots from HEK293T lysates showing indicated protein chase experiment. GFP is used as a loading control for transfection and Actin is used as an internal loading control. C Western blots showing indicated protein levels from TRIP12^+/+ and TRIP12^-/- HEK293T cells ± HA-ALK5. Phospho-SMAD2 is used as a control for HA-ALK5 expression. D qRT-PCR for indicated genes in HepG2 cells stably expressing sh-non template (NT) or shTRIP12. E Western blots showing indicated proteins after denaturing IP with HA-beads (ubiquitinated SMAD4) and Myc-beads (Control/SMAD4) from HEK293T cells of indicated genotypes. F Densitometric quantification from three independent experiment as in (E). Statistics were done by student’s t test. G Western blots showing SMURF2/SMAD4 interaction followed by SMAD4 IP in HEK293T cells of indicated genotypes. R1 and R2 are two different replicates in the same experiment. H Densitometric quantification from four independent experiments from (G). The dots represent a single SMURF2 densitometric value normalised to immunoprecipitated SMAD4 in the same experiment. I Western blots showing indicated proteins after denaturing IP with HA-beads (ubiquitinated SMAD4) and Myc-beads (Control/SMAD4) from HEK293T cells of indicated genotypes. J Densitometric quantification from two independent experiments as in (I). Statistics were done by student’s t test in (D, H, and F) and each dot in the plots represent mean of triplicates from three independent experiments unless otherwise stated. P = * < 0.05 and ** < 0.01.