Extended Data Figure 5: In vitro association of purified PP1Dis2 with PP2A-B55Pab1 and PP2A-B56Par1 complexes in a docking-site-dependent manner. | Nature

Extended Data Figure 5: In vitro association of purified PP1Dis2 with PP2A-B55Pab1 and PP2A-B56Par1 complexes in a docking-site-dependent manner.

From: A PP1–PP2A phosphatase relay controls mitotic progression

Extended Data Figure 5

ag, The tandem affinity purification steps used in Extended Data Fig. 10e were followed by immuno-affinity with antibodies directed against the HA epitope on B55Pab1.HA, B56Par1.HA fusion proteins or the unique sequence SQNWHMTPPRKNK in the C terminus of PP1Dis2(ref. 20) using anti-HA Affinity Matrix followed by elution with HA peptide, or Dynabeads A pre-loaded with PP1Dis2 antibodies20 followed by elution with the SQNWHMTPPRKNK peptide, respectively. b, Coomassie-stained 4–12% SDS–NUPAGE gradient gels. In each case the first two sample lanes show the purified wild-type and PDSN PP2A holoenzymes and the fifth lane shows the purified PP1Dis2 enzyme. The quantity of PP1Dis2 in the fifth lane was mixed with the quantity of each PP2A complex shown in lanes 1 and 2 before the PP1Dis2 was re-isolated from the mixture with beads that bore PP1Dis2 antibodies. This re-isolated PP1Dis2 (and any associated partner molecules) was then eluted from these beads with the SQNWHMTPPRKNK peptide and the eluted proteins run in lanes 3 and 4 of each gel. For both the B55Pab1 (left) and B56Par1 (right) PP2A holoenzymes, the wild type but not the PDSN complex bound to PP1Dis2. The numbered arrows indicate the lanes from which bands were excised for protein identification by mass spectrometry in c. c, Protein identification by mass spectrometry of the indicated bands from the lanes on the SDS–PAGE gels highlighted by the numbered arrows in b. emPAI scores45 show that the purified PP1Dis2 preparations used for the in vitro reconstitution reaction and run in lane 5 of the two gels in b also contained the conserved and well-characterized PP1 partner Sds22 (refs 46, 47). emPAI scores for the bands found in the reconstituted PP2A holoenzyme–PP1 complexes show that the predominant molecule identified in each complex corresponds with a known subunit of the PP2A complex, PP1Dis2 or Sds22. d, Western blotting to detect the indicated components in the complexes used for b and c. The epitope-tagged regulatory B55Pab1 and B56Par1 molecules were detected with antibodies against the HA tag, while the catalytic CPpa1 and CPpa2 and scaffolding APaa1 subunits of PP2A were detected with commercial antibodies. e, Western blots of gel filtration chromatography fractions of either the isolated PP2A holoenzyme complexes (top two blots) or the eluted PP2A holoenzyme/PP1 quaternary complexes (bottom two blots) corresponding to lanes 1 and 3 of the two SDS–NUPAGE gels of b. Note the very low level of PP2A-B55Pab1/PP1Dis2 and PP2A-B56Par1/PP1Dis2 quaternary complexes in the isolated PP2A holoenzyme preparations in the top two blots. Migration of the different complexes at the appropriate size suggests that the procedures used isolated correctly folded proteins. f, Phosphatase assays of the isolated enzymes used in be show that these molecules are active phosphatases and so the procedures used isolated functional, correctly folded complexes rather than denatured, inappropriately folded, proteins. Error bars show s.d. g, B56-Phos blots of the TAP- and HA-purified holoenzyme complex preparations used in c alongside a similarly purified PP2A-B56Par1.S378A holoenzyme complex. Biological replicates: for b, n = 3; for c and e, n = 1; d and g, n = 2; for f, n = 5.

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