Anti vsv m antibody

Anti-VSV M antibody was from KeraFAST, anti-p53 antibody (DO-1) and anti-p21 (F-5) were from Santa Cruz (#sc126 and sc-6246, respectively), anti-P-Erk (P-p44/42) and anti-Erk (p44/42) were both from Cell Signalling Technologies (#4370 and #4695, respectively), anti-Pan-Ras (OP40) was from Millipore, anti-actin was from MP Biomedicals, and anti-tubulin antibody was from Serotec. VSV-G antibody was a generous gift of Dr. I Ventoso (CBMSO, Madrid).

For analysis of SIV-Env and Gag or VSV-M incorporation into retro- and rhabdoviral particles, respectively, particles were purified via ultracentrifugation through a 20% sucrose cushion. The pellets were resuspended in SDS-PAGE sample buffer and analyzed by Western blotting. MLV-Gag was detected using an anti-MLV-p30 mouse antibody (Acris). SIV-Gag was detected using an anti-SIV-p27 mouse antibody (55-2F12). VSV-M detection was performed using an anti-VSV-M antibody (Kerafast). SIV-Env was detected using mouse monoclonal antibody DA6, which recognizes an epitope within gp120 (42 (link)). A horseradish peroxidase (HRP)-coupled goat anti-mouse IgG (H+L) antibody (Dianova) was used for detection employing a commercially available kit (Amersham). To analyze IFITM expression in sMAGI cells, an IFITM3-specific antibody was used (Proteintech).

Cells were washed in PBS, scraped in SDS-PAGE loading buffer and boiled for 5 min. Proteins of total extracts were separated by SDS-PAGE and transferred to nitrocellulose membrane. Western blotting was carried out using standard methods. All antibodies were used at a dilution of 1:1000 of the stock in blocking buffer (5% skim milk prepared in TBS-Tween). The membranes were incubated with the indicated antibodies, washed with TBS-Tween, and signals were detected by using chemiluminescence. Antibodies to PKR and eIF2α were purchased from Santa Cruz Biotechnology. Antibodies to phospho-eIF2α (Ser-51) and anti-SUMO2 were purchased from Life Technologies. Anti-VSV-M antibody was from KeraFAST. Anti-HA monoclonal antibody was purchased from Covance. Anti-actin antibody was from MP Biomedicals. Anti-phosphotyrosine antibody was from Cell Signaling. Anti-VSV-G antibody was a generous gift of Dr. I Ventoso.