Fig. 5

SYTL2 silencing impeded pseudopodia formation and promoted the proteasomal degradation of FSCN1 in a ubiquitin-independent manner. A Representative image of the Western blotting analysis of FSCN1 protein levels after SYTL2 knockdown or overexpression in DU145 and PC-3 cells. B PC-3 cells with or without SYTL2 knockdown were treated with cycloheximide (CHX) and harvested at different time points. The protein level of FSCN1 was analyzed by Western blotting analysis. C The relative protein expression level of FSCN1 was quantitatively analyzed by ImageJ and presented in the degradation curve. D PC-3 cells were treated with DMSO (control) or 10 μM MG132 for 6 h. MG132 impeded the alteration in FSCN1 protein expression levels mediated by SYTL2 knockdown (left panel) or SYTL2 overexpression (right panel). E To examine the alteration in the ubiquitination level of FSCN1 after changing SYTL2 expression, PC-3 cells transfected with vector or SYTL2 were treated with MG132 (10 μM). The cell lysates were immunoprecipitated with anti-FSCN1 antibodies, followed by immunoblotting using anti-ubiquitin. F–I Representative image of immunofluorescence in PC-3 (F) and DU145 (G) cells using the indicated reagents (DAPI shown in blue and phalloidin (shown in red arrow). The protrusions of cells, which represent the ability of pseudopodia formation, were quantitatively analyzed (H, I). Scale bars: 10 μm (white), *p < 0.05