Figure 3. Ectopic US28 expression induces PLC-β signaling in THP-1 cells.
(A) Transduced THP-1 cells were treated with 1 μg/ml of doxycycline for 24 hours. Immunoprecipitation and western blot with anti-flag antibody were used to detect US28 and mutant protein expression. Whole cell lysates were analyzed by western blot to detect actin expression. (B) The subcellular localization of US28 and US28-R129A were analyzed by immunostaining with anti-flag antibody and fluorescence microscopy. (C) Transduced THP-1 cells were labeled with 3H-myoinositol 1 day before induction, and treated with 1 μg/ml of doxycycline or vehicle (H2O) for 24 hours. Percentage of PLC-β induced IP conversion was calculated using the reading from column elution divided by the measure of total labelling. The graph represented four independent experiments. Statistical significance was determined by performing unpaired two-tailed Student’s t tests with GraphPad Prism® software. ΔN: N-terminal deletion US28, R129A: US28 with DRY box mutation. **:p<0.01, *: p<0.05, n.s.: not statistically significant.