Extended Data Figure 2: Association of PP1Dis2 with the PP2A regulatory subunits B56Par1 and B55Pab1. | Nature

Extended Data Figure 2: Association of PP1Dis2 with the PP2A regulatory subunits B56Par1 and B55Pab1.

From: A PP1–PP2A phosphatase relay controls mitotic progression

Extended Data Figure 2

ad, Immunoprecipitation reactions in which PP1Dis2 polyclonal antibodies precipitated and detected PP1Dis2, green fluorescent protein (GFP) antibodies precipitated and detected either PP1Dis2 or PP1Sds21 functional fusion proteins19 or 12CA5 monoclonal antibodies precipitated HA-tagged B55Pab1 and B56Par1 regulatory subunits of the PP2A phosphatase, as indicated. These blots establish that the association between PP1Dis2 and both B56Par1 and B55Pab1 was independent of PP1Sds21 function. No association was detected between PP1Sds21 and any PP2A regulatory subunit (including B56Par2 (data not shown)). b, c, Red asterisks indicate a non-specific band that was detected by the anti-GFP antibodies. d, No association was detected between B56Par2 and PP1Dis2, even when cell numbers in the precipitates were increased tenfold (numbers shown in red under the panel are x × 108 cells per ml) to enhance the sensitivity of detection and the B56par1+ gene encoding the B56Par1 subunit was deleted in an attempt to remove any competition from this primary B56 isoform. e, Expression of B56par2.PkC and B56par2. L482VPkC genes from the pINT41PkC integration vector32 was de-repressed by removal of thiamine. B56Par2 proteins (top) or PP1Dis2 (bottom) were precipitated with antibodies against the Pk epitope (top) or PP1Dis2 peptide (bottom), respectively, and these immunoprecipitation reactions were run alongside aliquots of the equivalent whole-cell extracts (WCE), before western blotting detected PP1Dis2 and B56Par2 fusion proteins. In both assays, the PP1-docking-site mutant in which the leucine of B56Par2 was replaced with the valine of B56Par1 (B56Par2.L482V) associated with PP1 whereas the wild-type B56Par2 protein did not. Biological replicates: for a, n = 6; for other panels, n = 2.

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