Mouse anti rac1

Mouse anti RAC1 (Millipore, 1:1000), rabbit anti RHOA (Cell Signaling, 1:1000), mouse anti CDC42 (Cytoskeleton, 1:1000), rabbit anti Glutathione-S-Transferase (GST; Santa Cruz, 1:2000) and rabbit anti EMERIN (Santa Cruz, Miami, FL, USA-254 1:1000) were used for western blotting.
The following antibodies were used for immunostainings: Mouse anti RAC1 (Millipore, 1:200), mouse anti active RAC1 (NewEast Biosciences, 1:200), rabbit anti C3 (Antibody Verify, 1:400), mouse anti Phospho-COFILIN (Santa Cruz, 1:200), mouse anti iododeoxyuridine (IdU)-B44 (BD Biosciences, 1:200, 347580).

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.

At 3 d after reperfusion, the rats were perfused with heparinized (10 U/ml) saline and then with 4% formaldehyde in phosphate buffered salin (PBS). The brain tissues were sectioned at 50 μm using a vibratome and stored at −20°C. The sections were washed three times with PBS, blocked with 5% bovine serum albumin (0.3% Triton X-100 in PBS) for 1 h and incubated overnight in primary antibodies. The primary antibodies used in this experiment were rabbit anti-CK2α (1:100; Millipore) and mouse anti-Rac1 (1:100; Millipore). The sections were washed three times with PBS and then incubated for 60 min with Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 594-conjugated goat anti-rabbit IgG. Sections were rinsed three times with PBS, mounted on glass slides and covered with mounting medium containing DAPI (Beyotime Institute of Biotechnology, Haimen, China).