Figure 6.

Effects of B4GALNT3 on β₁ integrin glycosylation and signaling. A: B4GALNT3 reexpression suppresses SK-N-SH cell migration toward collagen IV, fibronectin, and laminin (top panel). β₁ Integrin blocking antibody (Ab) (P4C10) significantly inhibits migration toward fibronectin (middle panel) and invasion (lower panel) of SK-N-SH cells. IgG is a control Ab. Results of three independent experiments are presented as mean ± SD. *P < 0.01. B: B4GALNT3 adds the LacdiNAc structure on β₁ integrin and affects its phosphorylation in SK-N-SH cells. Equal amounts of cell lysates from mock and B4GALNT3 transfectants were pulled down (PD) with WFA lectins and then Western blotted (WB) with an anti–β₁ integrin antibody (top set). Cell lysates were immunoprecipitated (IP) with an anti–β₁ integrin Ab and then were blotted with WFA lectins (middle set). B4GALNT3 decreases phosphorylation of β₁ integrin (bottom set). The β₁ integrins were IP with an anti–β₁ integrin antibody or a control IgG and then were immunoblotted with 4G10 or an anti–β₁ integrin antibody as indicated. C: B4GALNT3 decreases tyrosine phosphorylation of FAK, Src, and paxillin in SK-N-SH cells. The band intensity of pY397-FAK and pY418-Src was normalized to their total proteins. ^†The band intensity of pY118-paxillin and total paxillin was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This figure is the representative of three independent experiments. The signals were quantified by ImageQuant 5.1 and were normalized to their controls. D: β₁ Integrins are decorated with more LacdiNAc structures in B4GALNT3-stable transfectants than in mock transfectants of SH-SY5Y cells. B4GALNT3 overexpression was confirmed by WB analysis with anti-B4GALNT3 antibody. β₁ Integrins were PD with WFA and then immunoblotted with anti–β₁ integrin antibody. The signal was quantified by ImageQuant 5.1 and was normalized to their controls. E: B4GALNT3 decreases the phosphorylation (p) of FAK and paxillin in SH-SY5Y cells.