Figure 2.

Activation loop phosphorylation is important for the migration-promoting ability of ERK3 when re-expressed in H1299 cells with depletion of endogenous ERK3. A, generation of H1299 cells with stable expression of shCtrl) or shERK3. Knockdown of ERK3 was verified by Western blot analysis using an anti-ERK3 Ab. B, Transwell migration assay of stable H1299-shCtrl and H1299-shERK3 cells. Quantitated migration ability is presented as the number of migrated cells per field. Values in the bar graph represent mean ± S.E. (n = 6 fields). *, p < 0.001 (significantly different compared with shCtrl); Student's t test. C and D, H1299-shERK3 cells were transiently transfected with an empty vector or HA-tagged WT or mutant ERK3, as indicated. Two days post-transfection, cells were lysed and analyzed by Western blotting using an anti-ERK3 antibody and an anti-phospho-ERK3 (Ser¹⁸⁹) antibody (C). Cell migration ability was determined by Transwell migration assay and is presented as the number of migrated cells per field (D). Values in the bar graph represent mean ± S.E. (n ≥ 6 fields). **, p < 0.0001 (significantly different compared with empty vector); *, p < 0.05 (significantly different compared with empty vector); #, p < 0.0001 (significantly different compared with ERK3); one-way ANOVA. For all migration assays, representative images of migrated cells stained with crystal violet are shown below the bar graphs. Scale bars, 100 μm.