Anti gm130

U2OS cells plated on fibronectin-coated (15μg/ml) coverslip were either pre-extracted in 0.25% paraformaldehyde, PFA (16% stock solution Electron Microscopy Science) and 0.05% triton in cytoskeleton buffer (CB; 10 mM MES 6.1, 138 mM KCl, 3mM MgCl, 2 mM EGTA) for 1 min at 37°C, and fixed in 4% PFA in CB for 20 min, or fixed in 4% PFA in CB for 20 min at 37°C. After fixation, coverslips were permeabilized in 0.5% Triton X-100 in CB for 5 min, free aldehydes were reacted with 100 mM glycine, washed in TBS, and blocked in blocking solution (2% BSA TBS-T) for 1 hr. Cells were incubated with primary antibodies (anti-MARK2 (1:250, Abcam), anti-tubulin DM1A (1:500, Sigma), anti-GM130 (1:500 Cell Signaling), anti-S19-PMRLC (1:200, Cell Signaling), anti-myosin IIA (1:400; Sigma Aldrich,), anti-paxillin (1:500; BD Biosciences, San Jose, CA) or anti-MYPT1 (1:250, Abcam) diluted in blocking solution, washed 3 times 10 min each in TBS-T and then incubated with fluorophore-conjugated secondary antibodies (1:400; Jackson ImmunoResearch Laboratories) and Alexa Fluor 488 or 568 phalloidin (1:400; Invitrogen), washed again and mounted on slides with mounting media (Dako, Pathology Products, Carpinteria, CA), or in TBS supplemented with n-propylgalate for TIRF imaging.