pKO-integrin cells and WT and vinculin KO MEF cells were transfected using a similar protocol to the one previously described for eGFP-paxillin human fibroblasts (15). Cells were trypsinized, pelleted, resuspended in medium lacking FBS and penicillin/streptomycin, and counted. pKO-integrin cells (2 × 106) and WT (5 × 105) and vinculin KO cells were repelleted at 800 rpm for 10 min. P4 Nucleofector solution (82 μl) was added to 18 μl of P4 supplement in a 1.5-ml Eppendorf tube and used to resuspend the cell pellet. DNA for C-terminal eGFP-paxillin (Addgene, no.15233) cloned into the DNA 2.0 PiggyBac vector (~4 μg) was added to the cells and gently flicked before transferring to a Lonza nucleofection cuvette. Cuvettes were placed in a Lonza 4D-Nucleofector system and program C2167 (for MEFs) was used. Warm medium (500 μl) was added to the cuvette, and cells were transferred to a six-well plate with medium equilibrated at 37°C with 5% CO2 using a pipette bulb without pipetting up and down. Cells were selected with puromycin (1 to 2.0 μg/ml) 24 hours after transfection for 4 to 6 days.

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