Figure 4

Kinetics and stability of U _S 5-1 RNA. (A) Cultures of RSC in 25 cm² were mock infected (lane 1) or infected with 10 PFU of HSV-1(F) per cell and total RNA was extracted 1 (lane 2), 3 (lane 3), 6 (lane 4) or 9 h (lane 5) after infection and analyzes for the expression of U_S5-1 by northern blot using 0.7% formaldehyde agarose gel. The 28S ribosomal RNA is shown as the loading control. (B) Cultures of RSC in 25 cm² flasks were mock infected (lane 1) or infected with 10 PFU of HSV-1(F) per cell in the absence (lane 2) or presence (lane 3) of 100 μg/ml cycloheximide. Total RNA was extracted 9 h after infection and analyzed for the expression of U_S5-1 by northern using 0.7% formaldehyde agarose gel. The 28S ribosomal RNA is shown as the loading control. (C) Cultures of RSC in 25 cm² flasks were mock infected (lane 1) or infected with 10 PFU of HSV-1 (F) per cell in the absence (lane 2) or presence (lane 3) of 300 μg/ml of phosphonoacetate (PAA). Total RNA was extracted 9 h after infection and analyzed for the expression of U_S5-1 by northern blot using 0.7% formaldehyde agarose gel. The 28S ribosomal RNA is shown as the loading control. (D) Cultures of RSC in 25 cm² flasks were mock infected (lane 1) or infected with 10 PFU of HSV-1(F) per cell. At 6 h after infection actinomycin D (Act. D) was added to the concentration of 100 μg/ml of medium. RNA was extracted 0 (lane 2), 1 (lane 3), 2 (lane 4), 3 (lane 5) or 4 h (lane 6) after the addition of Act. D and analyzed for the expression of U_S5-1 by northern blot using 0.7% formaldehyde agarose gel. The 28S ribosomal RNA is shown as the loading control. (E) The amount of the U_S5-1 transcript in each experimental group in panel D was determined by measuring the density of each band on the membrane by UN-SCAN-IT densitometer (Silk Scientific, Inc., Orhem, UT). Relative amount of U_S5-1 transcript, expressed as the percent of the U_S5-1 transcript at time zero, was plotted as the function of time.