Profection calcium phosphate reagents

HEK293T cells were grown to 40–50% confluence in 12-well plates and co-transfected with an expression plasmid, a luciferase reporter plasmid, and pRL-TK (Renilla luciferase, internal control) using ProFection calcium phosphate reagents, according to the manufacturer’s instructions (Promega). Subsequently, 24–48 h post-transfection, the cells were collected and washed once with cold PBS. Passive lysis buffer (Promega) was subsequently added to the cells. After 15 min, supernatants were collected following centrifugation at 12,000 × g for 3 min, and relative luciferase activity was measured with the Dual-Luciferase Reporter Assay System according to the manufacturer’s instructions (Promega).

Luciferase assays were performed using the Dual-Luciferase Reporter Assay System kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol. In brief, 293T cells grown to 60%–70% confluence in 12-well plates and co-transfected with 500 ng of luciferase reporter plasmid containing the promoter regions of DLEU2 (luciferase reporter) and 100 ng of pRL-TK (Renilla luciferase, internal control) with using ProFection calcium phosphate reagents according to the manufacturer’s instruction (Promega). At 24 h post-transfection, the cells were infected with EV71 virus or Sendai virus (SeV) for the indicated durations. Mock-infected cells served as controls. Then, the cells were collected and washed once with cold phosphate-buffered saline (PBS). Passive lysis buffer (Promega) was then added to the cells. After 10–15 min, supernatants were collected following centrifugation at 12,000 × g for 30 s, and relative luciferase activity was measured with Dual-Luciferase Reporter Assay System according to the manufacturer’s instruction (Promega).

HEK293T cells were co-transfected with pcDNA-HA-CaMKIIα and pCAGGS-Flag-NS3 or pCAGGS-Flag-NS5 using ProFection calcium phosphate reagents (Promega). Subsequently, 24–36 h post-transfection, the cells were collected using western and IP buffer (Beyotime, Jiangsu, China) containing PMSF protease inhibitor according to the manufacturer’s instructions. Whole-cell extract (30 μL) was used as input; the remainder was divided into two equal parts and incubated with the Flag antibody and IgG, separately, at 4°C overnight with gentle rotation. Protein G magnetic beads (Invitrogen) were added into the lysates and incubated for 2–3 h according to the manufacturers’ instructions. Precipitates were washed five times with TBST buffer, and the proteins were eluted by boiling the beads for 5 min in 1× SDS sample buffer. Eluted proteins and input whole-cell extracts were analyzed by western blot.