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. Author manuscript; available in PMC: 2021 Oct 15.
Published in final edited form as: Mol Cell. 2020 Sep 22;80(2):345–358.e9. doi: 10.1016/j.molcel.2020.08.016

Figure 5. The Pol II CTD and Spt5 are in vitro and in vivo substrates of Integrator-PP2A.

Figure 5.

(A) Schematics of synthetic peptides used for in vitro phosphatase assays.

(B) Results of malachite green assay where peptides shown in panel A were incubated with Integrator-PP2A purified with anti-FLAG affinity resin from nuclear extracts expressing FLAG-PR65. Control is from S2 cells not expressing a FLAG-tagged protein. Representative colorimetric results are shown in the upper panel. Results from triplicate assays are quantified as picomoles of orthophosphate produced above negative control.

(C) Results of malachite green assay using Integrator-PP2A purified from cells expressing FLAG-IntS8-WT or FLAG-IntS8-WFEF/A mutant. The substrate used is CTD-Ser7P (n=3).

(D) Dose curves of Integrator-PP2A incubated with CTD-Ser7P and increasing amounts of PP2A inhibitors Calyculin A or Okadaic Acid (n=3).

(E) Western blot analysis of lysates from cells either mock-depleted or depleted of IntS8. Antibodies shown probe either endogenous proteins or are phospho-specific, as indicated.

(F) Western blot analysis of lysates from control or IntS8-depleted cells. Where indicated, cells were induced to express IntS8-WT or IntS8-WFEF/A mutant.