Ab31217

The bovine eIF4AI gene (GenBank accession no. 77735406) was synthesized by GenScript with the addition of an N-terminal FLAG tag and a C-terminal myc tag and cloned into pSNAP-tag (T7)-2 vector (New England BioLabs) using cut sites NdeI and NotI. Nucleotide sequences for 3C(wt), 3C(L127P), and 3C(C163A) with N-terminal FLAG tags were also cloned into pSNAP-tag (T7)-2 vector (New England BioLabs) using cut sites NdeI and NotI. Cell-free protein synthesis used a PURExpress in vitro protein synthesis kit (New England BioLabs) with the modification that two DNA plasmids were added in equimolar amounts. Western blots of cell-free synthesis products used anti-eIF4AI (ab31217, Abcam) antibody to detect eIF4AI and anti-DYKDDDDK (635691; TaKaRa) antibody to detect FLAG-tagged 3C.

Cells grown on cover glasses (VWR) were fixed with 3.7% formaldehyde (v/v) in PBS for 15 min at room temperature, immersed in methanol for 10 min at -20°C and blocked with 5% horse serum (Sigma) in PBS at 4°C overnight. Antibodies were diluted in blocking buffer as listed below and samples were incubated for 1 h with primary antibodies, followed by 30 min incubation with secondary antibodies at room temperature. cover glasses were mounted on glass slides using vinol mounting media [65 ] and imaged by confocal laser scanning microscopy using a Supercontinuum Confocal Leica TCS SP5 X equipped with a pulsed white light laser and a Leica HCX PL Apo 63x/1.40 oil objective. Images were processed using Adobe Photoshop. Settings for image acquisition and adjustment were kept constant for all samples for dsRNA signals. Settings for nsP3 and GFP were varied slightly between samples to compensate for strong differences in localised signal intensities. Primary antibodies: mouse anti-dsRNA (English and Scientific Consulting; 1:200), rabbit antiserum against SFV-nsP3 (1:500; [64 (link)]), rabbit anti-eIF2D (Abcam ab108218; 1:200), goat anti-eIF3B (Santa Cruz Biotechnology N-20 sc-16377; 1:200), rabbit anti-eIF4A (Abcam ab31217; 1:200). Secondary antibodies: Alexa Fluor 488 (Molecular Probes; 1:200), Alexa Fluor 568 (Molecular Probes; 1:1.000), Alexa Fluor 647 (Molecular Probes; 1:500).