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. 2013 Dec 26;289(7):4219–4232. doi: 10.1074/jbc.M113.518514

FIGURE 4.

FIGURE 4.

Specific oncogenic Ras variants selectively decrease PP5-ERK2, but not PP5-ERK1, interactions. A, HEK-293FT cells were transfected with HA-ERK1, HA-ERK2, or FLAG-PP5 alone or in combination and treated with nothing (Ø), 50 ng/ml EGF (5 min), 100 nm PMA (20 min), or an equivalent volume of DMSO (20 min) as a vehicle control. HEK-293FT cells transfected with FLAG-PP5 and HA-ERK1 or HA-ERK2 in combination with pcDNA3 (Vector) or HRasV12 were not treated prior to lysis. Western analysis of the FLAG immune complexes (FLAG IPs) and cell lysates was done using antibodies recognizing phospho-ERK1/2 (p-ERK1/2), Ras, HSP90, HA, and FLAG. Note that phospho-HA-ERK2 (p-HA-ERK2) and endogenous phospho-ERK1 (endog. p-ERK1) co-migrate. B, HEK-293FT cells transfected with (+) or without (−) HA-ERK2 or FLAG-PP5 were co-transfected with HRasV12, wild type HRas (HRasWT), HA-KRasV12, HA-KRasL61, or wild type HA-KRas (HA-KRasWT). FLAG IPs and cell lysates were analyzed by Western blot as in A. The data are representative of experiments performed four (A) and three (B) independent times with similar results.