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

This method was previously described (Xiang et al., 2013) . Briefly, HeLa cells grown in 60-mm dishes were incubated in methionine/ cysteine-free DMEM (Invitrogen) for 1 h and labeled with 1 ml of medium containing 250 µCi/ml Trans 35 S-Label [ 35 S] (MP Biomedicals, cat. no. 15100614) for 1 h. After being washed with PBS, cells were collected either immediately on ice or after incubation in 4 ml of complete growth medium containing 2 mM l-cysteine and lmethionine for the indicated time periods. Cells were lysed in 0.8 ml of lysis buffer (PBS containing 1 mM CaCl 2 , 0.5 mM MgCl 2 , 1% Triton X-100, protease inhibitor cocktail, and 1 µM pepstatin A) and centrifuged at 14,000 rpm for 15 min. The protein concentration of the supernatant was measured using a Bio-Rad protein assay (Bio-Rad Laboratories, cat. no. 5000006). The cell lysates (2 mg) were immunoprecipitated with 50 ng of the α5-integrin antibody BIIG2 and 60 ng of the β1-integrin antibody P5D2 overnight at 4°C. The antibodies were precipitated with 30 µl of protein G beads (Roche Diagnostics GmbH, Germany) at 4°C for 2 h. The beads were washed five times with lysis buffer, and the immunoisolated materials were eluted by boiling for 5 min in nonreducing SDS sample buffer. Integrins were resolved using 6.5% nonreducing SDS-PAGE followed by autoradiography.