Halt complete protease inhibitor cocktail

Whole cell lysates were generated by lysing cells in RIPA buffer (Thermo, Cat#89900) supplemented with Halt complete protease inhibitor cocktail (Thermo, Cat# 78429) and 1% n-dodecyl b-D-maltoside (DDM) (Thermo, Cat# BN2005). After 15 min on ice, lysates were sonicated using a Qsonica rod sonicator at 25% power for 3 s. Whole cell lysates were precleared by centrifugation at 15,000 × g for 10 min at 4°C. Protein concentrations were determined using the DC protein assay kit II (BioRad, Cat# 5000112) and standardized across all samples within an experiment. Protein lysates were reduced in Laemmli buffer (BioRad, Cat# 1610747) containing b-mercaptoethanol at 37°C for 30 min, but were not boiled to prevent aggregation of hydrophobic transmembrane regions. Equal amounts of protein were separated by SDS-PAGE and transferred to 0.2 μM nitrocellulose membranes (BioRad, Cat# 1620112). After blocking the membranes in 2% BSA in Tris-buffered saline (TBS)-Tween for 1 h at room temperature, they were incubated with primary antibodies (listed in the key resources table) at 4°C overnight and visualized either with HRP-conjugated secondary antibodies for chemiluminescence, or with up to 3 simultaneous fluorescent Alexa Fluor Plus-conjugated secondary antibodies. Images were acquired using the iBrightFL1000 system (Invitrogen).