Mouse anti g3bp1

For immunofluorescence microscopy, A549 cells were seeded on glass coverslips and cultured overnight prior to IAV-CA/07 infection at an MOI of 1 or mock infection. At the indicated times postinfection, cells were fixed with 4% paraformaldehyde and permeabilized with cold methanol as described previously (50 (link)). Cells were stained with goat polyclonal antibody to influenza A virus (catalog number ab20841; Abcam Inc., Toronto, ON, Canada), mouse anti-G3BP1 (catalog number 611126; BD Biosciences), mouse anti-PABP (catalog number 10E10; Santa Cruz), rabbit anti-TIAR (catalog number 8509; Santa Cruz), and anti-eIF3A (catalog number 3411; Cell Signaling), followed by Alexa-coupled donkey secondary antibodies (Thermo Fisher Scientific) at a 1:1,000 dilution, along with 5 ng/ml Hoechst dye (blue). Images were captured using a Zeiss AxioImager Z2 microscope. Bars represent 20 μm.

Rabbit anti-SINV antibody (used at 1 in 200) was a gift from Sondra Schlesinger, Washington University St Louis. Other antibodies were as listed below. They were used at the dilutions indicated and they were incubated either for 2 h at room temperature, or at 4 ℃ overnight. Rabbit anti- YBX1 (Abcam#ab12148, 1 in 300); rabbit anti-VCP (Cell Signaling Technologies, London, UK #2648); mouse anti-G3BP1 (BD, 611126, 1 in 200); goat anti-TIA1 (Santa Cruz sc-1751, 1 in 200); rabbit anti-PKR (Abcam#ab32052); rabbit anti-RIG-I (Abcam#ab45428, 1 in 200); rabbit anti-dsRed (Clontech 632496, 1 in 1000); mouse anti-ATG16L1 (MBL M150–3, 1 in 1000); mouse anti-actin (Sigma-Aldrich, Gillingham, Dorset, A5441, 1 in 7000); rabbit anti LC3 A/B (Cell Signalling#4108 1 in 1000). The antibody against phosphorylated eIF2 alpha was from Cell Signalling (#3398, 1 in 1000). Poly I:C (Roche) was transfected into cells with lipofectamine (Invitrogen) for four hours. Procarta mouse interferon alpha/interferon beta Luminex 2-plex was from Affimetrix (eBiosciences, ThermoFisher Scientific, UK).

Immunoprecipitation was performed as previously described [21 (link)] using mouse anti-GFP (Abcam, ab1218) and rabbit anti-SFV-nsP3 [57 (link)]. Immunoblotting was performed as previously described [4 ] using rabbit anti-SFV nsP3 [57 (link)], rabbit anti-CHIKV nsP3 [22 (link)], goat anti-actin (Santa Cruz #1616), rabbit G3BP-1 (Aviva Systems Biology, ARP37713) and rabbit anti-GFP (Abcam, ab290). Densitometry was performed as before [4 ].
For microscopy, BHK cells were processed as before [58 (link)]. Briefly, cells grown on coverslips were fixed with 3.7% (v/v) formaldehyde in PBS for 10 min at room temperature, followed by permeabilization with methanol at −20°C for 10 min, and blocking with 5% horse serum (Sigma) in PBS for 16 h at 4°C. Antibodies used were rabbit anti-SFV-nsP3 [57 (link)], rabbit anti-CHIKV nsP3 [22 (link)] and mouse anti-G3BP-1 (BD, 611126). Secondary antibodies were Alexa488-conjugated donkey anti-rabbit and Alexa555-conjugated donkey anti-mouse-IgG (Molecular Probes) and nuclei were stained using DRAQ5 (Thermo Scientific). Images were obtained by confocal laser-scanning microscopy using a Leica TCS SP5X microscope equipped with a super-continuum pulsed white laser and processed using Adobe Photoshop.