Anti pak2

GN and NB tissues were soaked in 10% paraformaldehyde (Sigma, St. Louis, MO, USA) at 4°C for 12 h and then dehydrated. After embedding, a rotary microtome (Leica, GER) was used to cut the tissue samples into 3.5 μm thick sections. The sections were immersed in a 42.5°C water bath and then mounted onto microscope slides (Citoglas, China). After deparaffinization and rehydration, the tissue sections were processed using reagents in an SABC kit (Bosterbio, China) according to instructions provided by the manufacturer. After processing, the mounted sections were incubated with anti-Ki67 (1 : 200, Abcam, UK) and anti-PAK2 (1 : 100, Abcam, UK) antibodies at 4°C for 12 h and then stained with reagents in a DAB kit (Bosterbio, China). The staining results were obtained using an inverted microscope (Nikon Eclipse TI-SR, Japan).

Protein samples were subjected to SDS-PAGE with appropriate concentrations, and then electro-transferred to nitrocellulose membranes (Millipore, MA). The membranes were blocked with 5% skimmed milk, washed and then incubated with primary antibodies overnight at 4 °C, followed by incubation with peroxidase-conjugated secondary antibody. Antibodies used were anti-actin (1:2000, Sigma), anti-EIF4EBP1 (1:500, Abnova), anti-phospho-EIF4EBP1 (T37) (1:500, Abnova), anti-PAK2 (1:500, Abcam) and anti-phospho-PAK2 (S141) (1:500, Abcam). Images were taken and estimated by densitometric analysis with Quantity One (Bio-Rad) and normalization with actin. Statistical analysis was performed with Student’s t-test and a threshold of p < 0.05 was defined as statistical significance cutoff.

Rabbit polyclonal anti-PAK2 (Cat# ab76293), anti-PLK1 (Cat# ab17056), and anti-BubR1 antibodies (Cat# ab254326) were obtained from Abcam (Cambridge, MA, USA); anti-Cdh1 antibody (Cat# sc-56312) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); mouse monoclonal α-tubulin-FITC antibody (Cat# F2168) from Sigma (St. Louis, MO, USA); anti-C-myc-antibody (Cat# 2278 S), HRP-conjugated rabbit (Cat #7074 S) and mouse (Cat# 7076 S) secondary antibodies were from Cell Signaling Technology (Danvers, MA, USA); GAPDH monoclonal antibody (Cat# 60004-1 g); Polyclonal Antibody Tubulin (11224-1-AP); Rabbit IgG (30000-0-AP) and Mouse IgG (B900620) were purchased from Proteintech (Wuhan, China); human anticentromere CREST antibody (Cat:#15-234) from Antibodies Incorporated (Davis, CA); Fluor 555 (Cat# A-11001) and 488 (Cat# A-21429) conjugated anti-mouse and anti-rabbit secondary antibodies from Invitrogen (Invitrogen, USA); and Cy5‐conjugated donkey anti‐human IgG (Cat #709-605-149) was purchased from Jackson Immuno Research Laboratory (West Grove, PA), Mouse anti-Rabbit IgG (Light Chain specific),HRP (BE0107-100) and Goat anti-Mouse IgG (Light Chain specific),HRP (BE0105-100) was purchased from Easybio (Beijing, China).