Anti myosin iia

Cells were seeded into a six-well dish on embryonic Day 0 and transfected with 25 μM myosin IIA siRNA (Dharmacon), Tpm 2.1 siRNA (Qiagen) or Tpm 3 siRNA (Dharmacon) using lipofectamine RNAiMAX (Invitrogen) on Day 1. Control cells were transfected with scrambled control siRNA (Dharmacon). Transfected cells were lysed in RIPA buffer (Sigma), and proteins extracted were separated by 4–20% SDS-polyacrylamide gel (Bio-rad) and transferred to PVDF membranes (Bio-rad) at 75 V for 2 h. Membranes were incubated with appropriate primary antibodies at 4 °C overnight: anti-myosin IIA (Sigma, dilution 1:1,000), anti-TM311 (Sigma, dilution 1:1,000), anti-TM γ9d (a gift from P. Gunning, dilution 1:1,000), anti-EGFR (CST, dilution 1:1,000), anti-HER2 (CST, dilution 1:1,000), anti-ROR2 (CST, dilution 1:1,000), anti-FLNA (abcam, dilution 1:1,000), anti-AXL (CST, dilution 1:1,000), anti-α-actinin (Sigma, dilution 1:1,000) and anti-α-tubulin (Sigma, dilution 1:3,000). Primary antibody binding was processed for ECL detection (Thermo Fisher Scientific) with appropriate horseradish peroxidase-conjugated secondary antibodies (Bio-rad). Uncropped immunoblot images are included in Supplementary Fig. 10.

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.