Fig. 4.

Alteration of ERK activity inhibits differentiation of ATDC5 cells. ATDC5 cells at 80% confluence were transfected with constructs expressing the following proteins: ERK2-WT (wild-type ERK2), ERK2–K52R (a kinase dead mutant), and MEK-R4F (constitutively active MEK1). Mock cells received only transfection reagent. After 24 h, cells were switched to medium containing insulin, and at d 3 after insulin addition, differentiation was assessed by RT-PCR for the markers Col2a1, Agc1,ColX, and Lox, with mock samples as calibrator; *, P < 0.001 (A). Simultaneous samples were collected and assessed for expression and activity status of ERK, MEK, and p38 by Western blotting (B). Note that exogenous ERK2 protein migrates above endogenous ERK2 because it contains a 3xFLAG peptide tag.