Rabbit anti cdc42

The fixed mouse sections were permeabilized with 0.1% PBS-Tx for 10 min, following which sections were washed twice with PBS for 10 min each. The sections were then blocked with 5% goat serum for 1 h. Next, the sections were incubated with rabbit anti-SLIT1 (1:25, ab129345, Abcam), mouse anti-ROBO2 (1:100, sc-376177, Santa Cruz Biotechnology), mouse anti-SRGAP1 (1:50, sc-81939, Santa Cruz Biotechnology) or rabbit anti-CDC42 (1:25, #2462, Cell Signaling Technology), overnight. The following day, the sections were washed with PBS thrice for 10 min each before incubation with secondary antibody for 1 h. Next, the sections were washed thrice with PBS thrice for 10 min each, and the nucleus was counterstained with DAPI. The coverslips were mounted using mounting medium (DAKO) and imaged using a confocal microscope (Olympus FV1000).

Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCL [pH 7.6], 150 mM NaCl, 1% NP40, 1% sodium deoxycholate [SDS], plus protease inhibitor cocktail set III [Calbiochem, Darmstadt, Germany]). Protein concentration of the lysates was quantified by using Bradford reagent (Applichem). Samples were separated by SDS-Page and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were probed with rabbit anti-ITK (ab32113, Abcam, Cambridge, United Kingdom, 1:2000), goat anti-GAPDH (EB06377, Everest Biotech, 1:20,000), rabbit anti-CDC42 (#24645, Cell Signaling, Cambridge, United Kingdom, 1:500) or mouse anti-RAC1 (88751, BD Biosciences, 1:500) followed by horseradish peroxidase-conjugated rabbit anti-mouse antibody or goat anti-rabbit antibody (GE Healthcare) or rabbit anti-goat antibody (R131HRP, Acris), and developed with ECL chemiluminescence reagents (GE Healthcare).

Tissues stored in liquid nitrogen were ground into powder. HEC-1A and Ishikawa cells were collected after 48 h of transfection. RIPA lysis buffer (Solarbio, Beijing, China) was added into the tissues powder and cells to extract total protein. The total protein concentration was investigated by BCA kit (Beyotime, Shanghai, China). For protein separation, 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with 10 μL of total protein samples. The separated protein was transferred onto polyvinylidene fluoride (PVDF) membrane. Then 5% skimmed milk was added to block protein for 1 h. Primary antibodies was used to probe protein for 12 h at 4 °C. The primary antibodies used in this study were as follows: rabbit anti-PAK1, anti-p-PAK1 and anti-VEGFA (1:1000, Abcam, Cambridgeshire, UK), rabbit anti-CDC42 and anti-GAPDH (1:1000, Cell Signaling, Beverly, MA, USA). The PVDF membrane was then treated by goat anti-rabbit secondary antibody (1:5000, Solarbio, Beijing, China) for 1 h at room temperature. The protein blots were visualized using enhanced chemiluminescence (ECL) system (Pierce Biotechnology, Rockford, IL, USA) according to the instructions. Image J software (NIH, Bethesda, MD, USA) was used for the blots intensity detection. GAPDH was the internal control.