Anti annexin a2

Exosome-like vesicles were lysed in 40 μL of lysis buffer (Promega) containing 1 μL of proteinase inhibitor cocktail (Sigma). The total protein concentration was measured using a Bradford assay containing Coomassie Plus protein reagent (Bio-Rad Laboratories) according to the manufacturer’s specifications. Equivalent amounts of total lysate were subjected to SDS-PAGE using 10% polyacrylamide gels. Proteins were electroblotted to polyvinylidene difluoride membrane (Millipore). The membranes were then blocked and incubated in anti-Annexin A2 (rabbit polyclonal; Abcam), Alpha-enolase (mouse monoclonal; Santa Cruz), Anexin A1 (mouse monoclonal; Abcam), and EpCAM (mouse monoclonal; Abcam). Alkaline phosphatase–conjugated anti-mouse or anti-rabbit IgGs were used as secondary antibodies (Bio-Rad) for detection. Then the membranes were incubated with Western Blotting Detection Reagents (Bio-Rad) according to the manufacturer’s instructions and exposed to autoradiography film.

Human and rhesus macaque astrocytes were pre-incubated for 1 h at 37°C with the following antibodies (50 μg/ml) against EV71 receptors: anti-CD162/PSGL-1 (BD Biosciences, San Jose, CA, USA), anti-annexinA2 (Abcam, Cambridge, UK), anti-DC-SIGN (Abcam), anti-SCARB2/LIMP2 (Novus Biologicals, Littleton, CO, USA), anti-FGF receptor 3 (FGFR3; Abcam), anti-CCR5 (GeneTex, San Antonio, TX, USA), and anti-CCR3 (GeneTex). The cells were then infected with EV71 (MOI = 0.05), washed twice with PBS and collected at different time points p.i. to determine the viral loads. This experiment has been repeated twice.
In the experiment using human purified complement, a low concentration of virus corresponding to an MOI of 0.005 was co-incubated with human purified complement (Cat. No. 204881, Merck Millipore) at concentrations of 5, 25, and 50 μg/ml (based on the physiological concentration in brain tissue; Barnum, 1995 (link); Jongen et al., 2000 (link)) or control buffer for 1 h, and then these mixtures were added to human astrocyte cultures for the observation of CPE. This experiment has been repeated twice.