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. Author manuscript; available in PMC: 2021 Apr 24.
Published in final edited form as: Cell Rep. 2021 Mar 30;34(13):108928. doi: 10.1016/j.celrep.2021.108928

Figure 5.

Figure 5.

MEK1/2 is a direct substrate of PP6 (A) PP6 complexes with WT or phosphatase inactive PPP6C (PD) were partially purified from HEK293T cells and incubated with phosphorylated MEK1 in vitro for the indicated times. Okadaic acid (OA, 100 nM) was added where indicated. Reactions were evaluated by immunoblot.

(B) In vitro phosphatase assays with phospho-ERK2 were carried out as in (A).

(C) Quantification of in vitro phosphatase assays in (A) and (B). Remaining phosphorylation is shown relative to the 30 min control reaction. Data are represented as mean ± SD. For MEK1 pSer218/pSer222, n = 4; for all other data, n = 3.

(D) HEK293T cells were co-transfected to express the indicated FLAG epitope-tagged PP6 subunit and untagged MEK1. Anti-FLAG immunoprecipitates and whole-cell lysates (WCLs) were evaluated by immunoblot for MEK.

(E) PPP6C+/+ and PPP6C−/− 501mel cells were transfected with non-targeting control siRNA or siRNA SMARTpools targeting PPP2CA and/or PPP2CB. Cells were lysed and evaluated by immunoblot for phosphorylated and total MEK.

(F) Quantification of the relative level of Phospho/Total MEK for PPP2CA/PPP2CB knockdown in PPP6C+/+ and PPP6C−/− cells in (E). MEK phosphorylation was normalized to PPP6C+/+, siRNA control. Data are represented as mean ± SD, n = 5. **p < 0.01, ***p < 0.001, unpaired t test.

(G) PPP6C+/+ and PPP6C−/− 501mel cells were transfected with non-targeting control siRNA or siRNA SMARTpools targeting PPP2CA and PPP2CB. Cells were lysed and evaluated by immunoblot for phosphorylated and total MEK. See also Figure S6.

(H) Quantification of the relative levels of Phospho/Total MEK for PPP2CA/PPP2CB knockdown in PPP6C+/+ and PPP6C−/− cells in (G). MEK phosphorylation was normalized to PPP6C+/+, siRNA Control. Data are represented as mean ± SD, n = 5. *p < 0.05, **p < 0.01, unpaired t test.