Fig. 7
ERK3 overexpression inhibited filopodia formation and decreased Rac1 phosphorylation in A431 cells. A431 cells were transduced with lentiviral empty vector pCDH or pCDH-HA-ERK3 (pCDH-ERK3) for 48 h followed by a immunofluorescent detection of ERK3 using a HA antibody and F-actin (Phalloidin staining), or b immunoblot analysis to confirm the overexpression of ERK3. Arrows in a indicate the filopodia. Immunoblot with β-actin was performed to confirm equivalent protein loading. c A431 cells were transiently transfected for two rounds with non-silencing control siRNA (NSC) or ΔNp63α siRNA (sip63) as indicated. Along with the second siRNA transfection, cells were also transfected with empty vector control (EV) or HA-tagged ERK3 plasmid as indicated. 48 h post-transfection, cells were harvested, followed by Western blotting analysis of ERK3, ΔNp63α, pRac1 and total Rac1 protein. β-actin was immunoblotted as the loading control. Numbers below the pRac1 blot indicate the relative signal of pRac1 normalized by total Rac1 protein level and the β-actin level under each condition. The pRac1 signal in Lane 1 was arbitrarily set as 1. d A simple model of the ΔNp63α/ERK3 axis suppressing Rac1 phosphorylation and cell migration. Representative western blots are shown as cropped gel images. Full length blots are presented in Additional File 6 (Figure S6)