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. Author manuscript; available in PMC: 2015 Oct 16.
Published in final edited form as: Nature. 2015 Jan 26;520(7547):378–382. doi: 10.1038/nature14044

Extended Data Fig. 9. TaPin1 changes c-Jun stability through the regulation of FBW7.

Extended Data Fig. 9

a. TaPin1 interacts with endogenous mouse FBW7. Protein extracts from NIH3T3 cells stably expressing HA-Flag-TaPin1 or HA-Flag-Control “Con” were used for HA immunoprecipitation, followed by immunoblot analysis using FBW7 and HA antibodies.

b. TaPin1 interacts with endogenous bovine FBW7 protein. Parasite-infected TBL3 and TpMD409 cell lysates were incubated with indicated GST-coupled beads, followed by immunoblot analysis using a FBW7 antibody.

c/d Analysis of indicated bovine gene expression by qPCR in TBL3 cells following Buparvaquone (Bup) or Juglone (Jug) treatment with or without TaPin1 WT or Mutant A53P. β-actin and H2A mRNAs were used for normalization (data represent 3 independent experiments - average ± sd, n=3).

e. siPin1 does not affect FBW7a or c-Jun protein levels. TBL3 were transiently transfected by siControl “siCon” or si-bovine-Pin1. Indicated protein levels were detected by Western blot analysis using specific antibodies. Actin was used as loading control.

f. Inhibition of TaPin1 by Buparvaquone/Juglone increased c-Jun ubiquitination and decreased FBW7 ubiquitination. Parasite-infected TBL3 cells (+/- Buparvaquone (Bup) or Juglone (Jug) - “Con” indicates Control) were incubated with MG132, followed by immunoprecipitation of endogenous c-Jun or FBW7 and immunoblot analysis with the indicated antibodies.

g. The effect of Buparvaquone on c-Jun ubiquitination was rescued by overexpression of TaPin1 Mutant A53P. Parasite-infected TBL3 cells (+/- Buparvaquone (Bup) or Juglone (Jug) - “Con” indicates Control) were transfected with TaPin1 WT, TaPin1 Mutant A53P or empty vector “Con” and then treated with MG132, followed by immunoprecipitation of endogenous c-Jun and immunoblot analysis with the indicated antibodies.

h. Measure of c-Myc, KLF5 and activated Notch 1 protein levels in TBL3 upon Buparvaquone (Bup) treatment. “Con” indicates Control. Tubulin was used as loading control.

i. Measure of c-Myc, KLF5 and activated Notch 1 protein levels in TBL3 upon FBW7α ectopic expression. “Con” indicates Control = transfection with the appropriate empty vector. Tubulin was used as loading control.

All the results in a, b, e, f, g, h and i are representatives of 3 independent experiments.