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. 2010 Sep 24;285(48):37650–37662. doi: 10.1074/jbc.M110.138818

FIGURE 4.

FIGURE 4.

PMA- and EGF-stimulated phosphorylation of β4 is dependent on ERK1/2 activation. A, PA-JEB/β4 keratinocytes, starved overnight in growth factor-free medium and pretreated for 1 h with U0126, PD98059 or SB203580, were left unstimulated or stimulated with PMA or EGF for 10 min. Cell lysates were analyzed by immunoblotting with antibodies specific to phosphorylated β4 (Ser-1356 and Ser-1364), total β4, phospho-ERK1/2, and total ERK1/2 (B) COS-7 cells, transiently expressing β4 alone or together with ERK1 or a dominant-negative version of ERK1, were starved overnight in growth factor-free medium, and then left unstimulated or stimulated with EGF for 10 min. Phosphorylated β4 (Ser-1356 and Ser-1364), total β4, phospho-ERK1/2 and total ERK1/2 were detected by immunoblotting. C, PA-JEB keratinocytes, stably expressing wild-type β4 (FAFP) or β4 with a mutated ERK docking site (AAAP), were starved overnight in growth factor-free medium, and then left unstimulated or stimulated with EGF or PMA for 10 min. Phosphorylated β4 (Ser-1356 and Ser-1364), total β4, phospho-ERK1/2, and total ERK1/2 were detected by immunoblotting. The graphs show the fold increase in phosphorylation of Ser-1356 and Ser-1364 after EGF or PMA stimulation. The intensity of the bands corresponding to phosphorylated β4 were related to that of total β4 using ImageJ.