Irdye 680 and 800 labeled secondary antibodies

CK cells were mock infected or infected with the indicated viruses, and at 24 h postinfection, cells were scraped into the culture medium and pelleted. Cell pellets were washed once with cold PBS and then lysed in radioimmunoprecipitation assay buffer (150 mM NaCl, 50 mM Tris [pH 7.4], 1% Igepal, 0.25% sodium deoxycholate, 1 mM sodium orthovanadate, 1× protease inhibitor cocktail) on ice for 20 min. Cell lysates were mixed with 4× solubilization buffer (SB; Bio-Rad) to give a 1× final concentration and denatured at 95°C for 3 min. Proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. After blocking overnight in 5% milk in PBS-Tween 20, the membrane was labeled with anti-S2 (catalog number 26.1; Thermo Fisher) and anti-beta-actin (Abcam), followed by IRDye 680- and 800-labeled secondary antibodies (Li-Cor). Blots were imaged using an Odyssey CLx system (Li-Cor).

The following antibodies were used in this study: β-actin (A1978; Sigma; Dilution 1:5000), V5 (V8012; Sigma; Dilution 1:1000), EV71-VP1 (MAB1255-M05; Abnova; Dilution:1:1000), and SETD3 (ab176582; Abcam; Dilution 1:2000). IRDye 680- and 800-labeled secondary antibodies were purchased from LI-COR Biosciences (926-68020, 926-32211, 926-68073; Dilution: 1:5000-1:20000). Anti-EV71 2A antibody was generated in rabbits using recombinant protein as the immunogen (Dilution 1:5000). Latrunculin A (L5163) was purchased from Sigma.