Mouse anti cdc42

For Western immunoblotting analysis, infected neurons were collected in 1 × LDS loading buffer (Invitrogen) and boiled for 10 min. Proteins were separated by electrophoresis in 12% NuPAGE gels (Invitrogen). To better detect ATF4, the gels were run a longer time (80–100 min) to separate ATF4 from a closely migrating non-specific band. The following primary antibodies were used: rabbit anti-ATF4 (1:250, PRF&L), mouse anti-RhoA (1:1000; Cytoskeleton), mouse anti-RhoA-GTP (1:500; Santa Cruz), mouse anti-Rac1 (1:5000; Millipore), mouse anti-Cdc42 (1:500; BD), rabbit anti-pLIMK1/2 (1:500; Santa Cruz), and mouse anti-GAPDH (1:2000; Imgenex).
Immunocytochemistry was performed on the primary hippocampal neurons cultured on 15-mm cover glasses. Neurons were fixed with 4% PFA for 15 min and blocked with 5% BSA. The following primary antibodies were used: rabbit anti-GluR1 (1:200; Cell Signaling), rabbit anti-PSD-95 (1:200; Cell Signaling).
For DiOlistic labeling, the Helios gene gun system (Bio-Rad) was used according to the manufacturer's instructions. Tungsten particles (1.1 μm, Bio-Rad) coated with Dil (Invitrogen), which defines the neuronal architecture in red, were delivered into fixed neurons on coverslips or brain sections. Coverslips were mounted in ProLong Gold antifade reagent (Invitrogen) and imaged the next day.

The following primary antibodies were used: rabbit anti-FSCN1 (Abcam, ab126772; IF 1:200, WB 1:1000, IP 1:100), mouse anti-GAPDH (Abcam, ab8245; WB 1:1000), mouse anti-F-actin (Abcam, ab205; WB 1:1000), rabbit anti-YAP (Cell Signaling Technology, 14074; IF 1:200, WB 1:1000), rabbit anti-p-YAP (Ser127) (Cell Signaling Technology, 4911; WB 1:1000), mouse anti-CDC42 (BD Biosciences, 610929; WB 1:500), rabbit anti-VEGFR2 (Cell Signaling Technology, 2479; WB 1:1000), rabbit anti-p-VEGFR2 (Tyr1175) (Cell Signaling Technology, 2478; WB 1:1000), goat anti-DLL4 (R&D Systems, AF1389; IF 1:200), and mouse anti-β‐actin (Millipore, A5441; WB 1:1000). The following secondary antibodies were used: goat anti-mouse HRP-conjugated antibodies (Beyotime, A0216; 1:1000) and goat anti-rabbit HRP-conjugated antibodies (Beyotime, A0208; 1:1000) were applied for WB, Alexa Fluor™ Plus 488 Anti-mouse IgG (H + L) (Abcam, ab150077; IF 1:400), and DyLight 405-AffiniPure Donkey Anti-goat IgG (H + L) (Jackson ImmunoResearch,705–475-147,1:400) was applied for immunofluorescence.

Western blot analysis was performed by using a standard procedure. In brief, equal amounts of protein (30 μg) were loaded to SDS PAGE, which was blotted to nitrocellulose (NC). The membranes were blocked 30 min at room temperature with 5% milk powder in Tris-buffered saline and Tween 20 (TBST). After this the membranes were incubated with the respective antibodies in 5% milk/TBST solution overnight at 4 °C. The antibodies used were: rabbit anti-Diaph2 (Sigma-Aldrich Cat# HPA005647), rabbit anti-ß-tubulin (Sigma-Aldrich Cat# T4026), mouse anti-actin (Sigma Cat# A 2066), rabbit anti-Hsc70 (Santa Cruz Biotechnology Cat# sc-7298), rabbit anti-detyrosinated ß-tubulin (abcam Cat# ab48389), mouse anti-Cdc42 (BD Bioscience Cat# 610928). Secondary antibodies against mouse (1:10.000; abcam Cat# ab205719) or rabbit (1:10.000; abcam Cat# ab205718) were diluted in TBST and incubation was performed for 1 h at room temperature. After visualization by chemiluminescence reagent (Amersham ECL Prime Western Blotting Detection Reagent, GE Healthcare Bio-Sciences) and ImageQuant LAS 4000 (GE Healthcare), band intensities were quantified by ImageJ (NIH National Institutes of Health) and calculated as percent intensity of the specific control sample.