Pei max polyethylenimine reagent

Jeg3 cells originally obtained from ATCC.org (Cat# Htb-36) were maintained in a humidified incubator at 37°C and 5% CO₂. Jeg3 cells were maintained in 1× MEM (Thermo Fisher Scientific; Cat# 10370088) with penicillin/streptomycin (Thermo Fisher Scientific; Cat# 15070063), 10% fetal bovine serum (FBS; Thermo Fisher Scientific; Cat# 26140079), and GlutaMAX (Thermo Fisher Scientific; Cat# 35050061). Cultures were maintained on Corning 100 × 20 mm Petri-style TC-treated culture dishes (Cat# 430167). Transient transfections were accomplished using a PEI MAX polyethylenimine reagent (Polysciences; Cat# 24765). DLD-1 cells originally obtained from ATCC.org (Cat# CCL-221) were handled identically with the exception of using DMEM media. All cell lines were checked for mycoplasma monthly by DAPI staining.

To determine the interaction between ARHGAP18 and ezrin, pulldown assays were performed using cell lysate from Jeg3 wildtype and Jeg3 LOK^-/-SLK^-/- cells. Transfections were performed using PEI MAX polyethylenimine reagent (Polysciences; Cat# 24765). After transfection, cells were washed with phosphate-buffered saline (PBS) and harvested with lysis buffer (25 mM Tris, 5% glycerol, 150 mM NaCl, 50 mM NaF, 0.1 mM Na₃VO₄, 10mM βGP, 0.2% Triton X-100, 250 mM calyculin A, 1 mM DTT, 1× cOmplete Protease Inhibitor Cocktail [Roche; Cat# 11836153001]) by scraping. Lysates were then sonicated, and any insoluble material was centrifuged at 8000 × g for 10 min at 4°C. Before incubating with the cell lysates, SUMO-ARHGAP18 NiNTA or M2-Flag resin beads were equilibrated and washed into lysis buffer. The sample of the supernatant was taken for input, then the rest was added to the SUMO-ARHGAP18 NiNTA or M2-Flag beads and nutated for 3 hr at 4°C. After incubation, the beads were pelleted and washed four times before boiling in 40 µL 2× Laemmli sample buffer.