Immunoblotting was performed as previously described.15 (link) Briefly, proteins in cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF (polyvinylidene difluoride) membranes (Millipore, Bedford, MA, USA). The membranes were blocked by incubating with a Tris/saline/0.1% Tween-20 solution (TBS-T) containing 5% nonfat dry milk for 1 hour at room temperature and incubated with a primary antibody overnight at 4°C. Membranes were incubated with species-appropriate HRP-conjugated secondary antibodies for 1 hour and visualized using an enhanced chemiluminescence (ECL) kit (Santa Cruz Biotechnology), with detection by exposure to X-ray film (Kodak, Rochester, NY, USA) or Imagelab system (Bio-rad). Immunoprecipitation assays were performed as previously described.16 (link) Protein interactions and ubiquitination modifications were analyzed by incubating anti-antithrombin, anti-GFP, or anti-FLAG antibody with a Dynabeads Antibody Coupling Kit (Life Technologies, Invitrogen, CA) according to the manufacturer’s instructions, after which cell lysates were incubated with beads for 3h at room temperature. Precipitated proteins were washed three times with PBS containing 0.1% Tween-20 (PBS-T), eluted using Blue Loading Buffer Pack (Cell Signaling Technology), resolved by SDS-PAGE, and immunoblotted.