Figure 2.

EV71 infection induced an intracellular redistribution of GRP78/BiP. (A, B and E) Cytoplasmic/microsomal localization of GRP78/BiP during viral infection. Homogenates extracted from mock, EV71-infected (MOI of 10, 6 h p.i.) or DTT-treated (2.5 mM, 6 h) RD (A and E) or HEK293T cells (B) were subjected to cytosolic/microsomal fractionations. After centrifugation, fractionates were resolved on SDS–PAGE followed by western blotting with antibodies against the indicated proteins. (C) Cell surface distribution of GRP78/BiP in EV71-infected RD cells. RD cells were infected with EV71 at an MOI of 10, and the membrane proteins were prepared using a Plasma Membrane Protein Extraction Kit at 6 h p.i. Annexin II and GAPDH served as markers for the plasma membrane and cytosol fractions, respectively. (D) Immunoblot analysis of cell lysates and culture medium obtained from mock-infected or EV71-infected cultures. Thirty micrograms of lysates or Amicon filter unit-concentrated supernatant was subjected to SDS–PAGE and western blotting. Calnexin, GAPDH and β-actin were used as markers for microsomes, cytoplasm and internal controls, respectively. The results are representative of three independent experiments. An asterisk marks nonspecific bands.