GRASP depletion–mediated Golgi destruction decreases cell adhesion and migration via the reduction of a5ß1 integrin

Ahat, Erpan; Xiang, Yi; Zhang, Xiaoyan; Bekier, Michael E.; Wang, Yanzhuang

All procedures were performed on ice or at 4°C. Cells grown on 15-cm plates were incubated on ice for 20 min, washed three times with PBS, treated with 6 ml of 0.5 mg/ml NHS-SS-biotin (Fisher, cat. no. P121331) in PBS for 20 min, and quenched by 100 mM glycine/ PBS for 10 min. After three washes with PBS, cells were lysed in RIPA buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, 1 mM EDTA, 0.5% sodium orthovanadate, 0.1% sodium fluoride, and 1× protease inhibitor cocktail [Bimake, cat. no. B14001]). Cell lysates were adjusted to 5.86 mg/ml in lysis buffer, and 3-mg samples were incubated with 50 µl of streptavidin-agarose beads (GE Healthcare, at 4°C overnight. After being washed, beads were boiled in SDS sample buffer with 100 mM DTT, and bound proteins were analyzed by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, and blotted with α5-integrin (Cell Signaling, cat. no. 4705), β1-integrin (Cell Signaling, cat. no. 4706), TfR (Invitrogen, , and β-actin (Proteintech, cat. no. 66009-1-Ig) antibodies.