Eif4g1

We performed PLA as described before.^{26 (link)} Briefly: 2×10e6 cells were treated for 4 h as indicated. Cells were washed with 1× phosphate buffered saline (PBS) (Corning, NY) and fixed with 4% paraformaldehyde (Thermo Fisher Scientific). CometSlides (Trevigen, Gaithersburg, MD) were coated with Poly-L-Lysine 0.1% solution (Sigma–Aldrich (SA), St. Louis, MO), and cells were allowed to adhere. We followed the protocol of Duolink PLA;^{27 (link)} briefly: Cells were blocked using Duolink blocking solution, followed by probing with primary antibodies for eIF4G1 (Cell signaling Technologies, Danvers, MA, Cat. #2858, 1:200 dilution) and eIF4E (BD Biosciences, San Diego, CA, Cat. #610269, 2.5 µg/ml final). Next, cells were incubated with Duolink In Situ PLA Probe Anti-Rabbit PLUS (Cat. # DUO92002) and Duolink In Situ PLA Probe Anti-Mouse (Cat. # DUO92004) and allowed to ligate using ligation mix. Next, amplification and washes were performed as instructed and the slides were mounted using media containing DAPI. Slides were imaged using Leica TCS SP8 confocal microscope. Signal obtained was quantified using ImageJ software, and normalised to the number of cells per field (using DAPI nuclei staining). Images shown indicate the signal (Orange Duolink^TM) and nuclei for each field imaged, while graphs presented indicate ratio values of signal per cell in each field imaged.

HeLa cell protein was isolated using 1X PBS lysis buffer (1X PBS, 0.5 mM EDTA, Triton X-100, and protease inhibitor mixture) and mixed with sample buffer. The protein was separated on a 10% SDS-PAGE system and then carried to nitrocellulose membranes. The membranes were probed with the anti-CVB3 VP1 antibody (ThermoFisher Scientific, Cambridge, MA, USA), eIF4G1, BAX, BAD, phosphor ERK, total ERK, cleaved caspase3, and GAPDH antibodies (Cell Signaling, Danvers, MA, USA) and refrigerated for 18 h at 4 °C. Antibody binding was detected using the ECL system (Intron Biotech, Inc., Seongnam-si, Republic of Korea) and the results were quantified using NIH-ImageJ1.45s software (Wayne Rasband, MA, USA).