Immunoblotting of Cellular Proteins

Cells were collected in a sodium dodecyl sulfate (SDS) lysis buffer. After sonication, the proteins were subjected to electrophoresis in a 5–20% SDS–polyacrylamide gel, and then transferred onto a polyvinylidene difluoride membrane (ATTO), followed by overnight blocking with 5% skimmed milk in phosphate-buffered saline with Tween 20 (Bio-Rad, Hercules, CA, USA). The membrane was probed with antibodies specific to MAP2K3 (Cell Signaling Technology, Boston, MA, USA), GAPDH (Santa Cruz), and/or β-tubulin (Abcam K.K., Tokyo, Japan). After washing the membrane, it was incubated with secondary horseradish peroxidase-conjugated antibodies for an hour. The signals were detected with enhanced chemiluminescence (GE Healthcare, Tokyo, Japan) and scanned with an image analyzer LAS-4000 mini (Fuji Film, Tokyo, Japan), as previously described [13 (link)].