Iblot blotting system

The purified proteins were analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS–PAGE) using 4–12% Bis‐Tris NuPAGE gels (Invitrogen). Prior to SDS–PAGE analysis, protein samples were heated in the presence of 10 mM DTT at 90°C for 10 min. For Western blot analysis, proteins from SDS–PAGE gels were transferred onto a nitrocellulose membrane using iBlot blotting system (Thermo Scientific). Membranes were blocked for 1 h in TBST (10 mM Tris–HCl, 150 mM NaCl, and 0.05% Tween 20, pH 8) containing 2% (w/v) biotin‐free bovine serum albumin (BSA). The Strep‐II‐tagged proteins were immunodetected using the alkaline phosphatase‐conjugated Strep‐Tactin (IBA) and the color reaction was developed with 5‐bromo‐4‐chloro‐3‐indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT). The Blue native PAGE was performed using the Novex 4–16% Bis‐Tris gels (Invitrogen) according to the manufacturer's instructions.

Cells were washed with PBS and then lysed in lysis buffer (50 mM Hepes, pH 7.2, 150 mM NaCl, 50 mM NaF, 1 mM Na₃VO₄, 10% glycerol, 1% Triton X-100). The lysates were then centrifuged at 12,000 × g for 15 min at 4 °C. The proteins were denatured in Laemmli sample buffer (5 min at 95 °C), separated by SDS/PAGE, and transferred to nitrocellulose membranes using the iBlot blotting system (Invitrogen). Membranes were blocked in PBS with Tween 20 (PBST) containing 5% BSA for 30 min before being incubated with antibodies for 3 h. VIM protein (Fitzgerald) and bone marrow lysates from C57BL/6J or VIM-deficient mice were used for identification. After washing the membranes several times with PBST, the blots were incubated with B1 Ab or horseradish peroxidase-conjugated anti-VIM or anti–β-actin antibody for 1 h. The membranes were then washed with PBST and developed by ECL. Phosphorylation was performed with phospho-AKT, ERK, p38, and VIM S38 (Cell Signaling Technology).