Fig 1.

Phosphorylation of BRN2 affects cell migration and proliferation in cellulo. (A) Analysis of BRN2 phosphorylation profile. BRN2TS and BRN2AA were produced in bacteria, purified, and incubated with [γ-³²P]ATP in the presence of purified PKA. The proteins were analyzed by SDS-PAGE. Com, Coomassie staining. (B) In cellulo motility of murine melan-a melanocytes. Cells were transfected with GFP-coupled BRN2TS and BRN2AA expression vectors. The empty GFP expression vector (Mock) was used as a control. Forty-eight hours after transfection, the cells were subjected to a single-cell migration assay using two-dimensional video microscopy for 12 h and cell motility was determined. Each experiment was performed at least three times. The migration speeds were calculated as means ± standard deviations using Excel (Microsoft). Student's t test was used to compare migration speeds between groups. *, P < 0.01; ***, P < 0.001. (C) The cell proliferation rates of Lyse melanoma cells after 48 h of BRN2TS and BRN2AA expression were estimated by following BrdU incorporation into transfected cells over 2 h. Experiments were performed at least three times. The proliferation rates were calculated as means ± standard deviations using Excel (Microsoft). Student's t test was used to compare migration speeds. *, P < 0.01; **, P < 0.005. Similar results were obtained with all of the cell lines tested.