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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 4. PPP6C regulates ERK signaling via MEK1/2.

Figure 4.

(A) shCTRL, shPPP6C-1, and shPPP6C-2-expressing 501mel cells were transfected with non-targeting control siRNA or siRNAs directed to ARAF, BRAF, or CRAF as indicated. Cells were lysed and assessed by immunoblot for phosphorylated and total MEK and ERK.

(B) Quantification of the relative levels of Phospho/Total MEK and ERK from (A) was normalized to shCTRL, siCONTROL. Data are represented as mean ± SD, n = 3.

(C) BRAF was immunoprecipitated from 501mel cells expressing shCTRL, shPPP6C-1, or shPPP6C and evaluated in vitro in kinase assays on MEK1 over the indicated time course. Vemurafenib (1 μM) was added to negative control reactions. Reactions were evaluated by immunoblot.

(D) 501mel cells expressing shCTRL, shPPP6C-1, and shPPP6C-2 were lysed and assessed by immunoblot for MEK phosphorylation at Ser218/Ser222, Thr286, and Ser298. Non-specific cross-reacting bands in the pThr286 and pSer298 blots are indicated with an asterisk.

(E) Quantification of the relative levels of Phospho/Total MEK from (D) was normalized to shCTRL. Data are represented as mean ± SD, n = 3.

(F) PPP6C+/+ and PPP6C−/− 501mel cell lines were transiently transfected to express His epitope-tagged MEK1 or MEK2. Cell lysates were analyzed by immunoblot for phosphorylated and total MEK. Open arrows indicate ectopically expressed His-tagged MEK1/2, and solid arrows indicate endogenous MEK1/2. See also Figure S5.