Anti kif2a

Total protein (tissue samples and cells) was extracted with the RIPA buffer containing a protease inhibitor cocktail (Roche, Basel, Switzerland). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (10%, SDS-PAGE, Sangon Biotech, Shanghai, China) was conducted to isolate total protein. Next, the isolated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Roche) and then blocked with tris buffered saline tween (TBST) buffer containing 5% skim milk. After washing with TBST, the membranes were incubated with primary antibodies at 4 °C overnight, including anti-Cleaved PARP (#9541, 1:1000), anti-B-cell lymphoma/leukaemia-2 (Bcl-2) (#4223, 1:1000), anti-Bcl-2-associated x (Bax) (#2772, 1:1000), anti-KIF2A (ab197988, 1:200, Abcam, Cambridge, MA, USA), and anti-β-actin (#4967, 1:1000). Subsequently, the membranes were incubated with goat anti-rabbit IgG (#7077, 1:2000). Protein bands were visualized with an ImmunoStar LD (Wako Pure Chemical, Osaka, Japan). β-actin was used as a loading control. All antibodies were purchased from Cell Signaling Technology (Santa Cruz, California, USA), except for KIF2A.

Total proteins prepared from DCs were separated on a SDS polyacrylamide gel (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Amersham Pharmacia Biotech). Following blocking with 5% nonfat milk in TBS-T (Tris buffered saline plus 0.05% Tween-20), the membrane was incubated with anti-MT1-MMP (Millipore Biosciences), anti-KIF2A (Abcam) or anti-HIF-1α (Abcam) at 4 °C overnight. Then, the membrane was washed 4 times, 5 min each in TBS-T before incubating with horseradish peroxidase (HRP)-conjugated goat anti- rabbit or mouse IgG (Jackson Immuno Research Laboratory). Anti-β-actin mAb (Abcam) was used as an internal control. Protein bands were visualized with enhanced chemiluminescence detection system (ECL, Amersham Pharmacia Biotech) following the manufacturer’s instructions.