Figure 5

Down-regulation of Hsp16 activation by Vpr. (A) Expression of hsp16 monitored by GFP-Hsp16 fusion protein expression. For heat shock treatment, vpr gene expression was first induced at 30ºC for 21 hours and then cultures were treated with acute heat shock (Acute HS) at 45ºC for 15 min (middle columns) or exposed to constant 36°C (right columns). The level of Hsp16 expression was examined 2 hrs after the heat shock, i.e., 23 hrs after vpr gene induction (GI). (B) Comparison of the Hsp16 protein levels between acute heat shock and constant heat treatment shown by Western blot analysis. The hsp16-expressing vpr (RE007) and vpr’ (RE076) cells were collected at the same time as in (A), i.e., 23 hrs after vpr gene induction. a, Hsp16 protein levels under acute heat shock conditions. Acute heat shock (45ºC for 15 min) was used to transiently activate Hsp16, and the Vpr effect was measured 2 hrs after the heat shock. b, Hsp16 protein levels under constant and prolonged high temperature at 36°C. The effect of Vpr on Hsp16 was measured 48 hrs after cell culturing at 36ºC, which normally induces constant elevation of Hsp16. Lane 1 shows wild type SP223 cells without plasmid (Cell Ctr); lane 2 shows SP223 cells carrying an empty plasmid (Plasmid Ctr); Ctr, control; GI, gene induction, +, vpr-on; -, vpr-off. LC, a protein band that non-specifically reacted to the antibody and was used as a protein loading control.