Expression of Zf9 by BAECs.
(A) and (B) Time course in induction by serum and PMA. After a 5-hour preincubation with serum-free medium confluent BAEC cultures in either 15-cm dishes or 6-cm dishes were incubated with 10% FCS-containing medium for the indicated times, in the absence and presence of 10 ng/mL PMA. Cell lysate was prepared from each 15-cm dish, total RNA was isolated, and 30 μg of each RNA was used for detection of zf9mRNA in Northern blotting (panel A). Cells on each 6-cm dish were scraped into 60 μL of sodium dodecyl sulfate (SDS) sample buffer, and rapidly passed 20 times through a 17-gauge needle to shear nucleic acids. A portion (30 μL) of each cell lysate was immediately subjected to SDS-polyacrylamide gel electrophoresis on 13% resolving gel without reducing conditions, and changes in Zf9 protein levels were analyzed by Western blotting as described in “Materials and Methods” (panel B). Lane 1, control; lanes 2-6, without PMA; lanes 7-11, with PMA. Each experiment was repeated 5 times with similar results and representative results are shown. (C) and (D) Disappearance of Zf9 expression by BAECs after depletion of serum. Following a 5-hour incubation with 10% serum ± 10 ng/mL PMA, BAECs were then incubated with serum-free medium ± 10 ng/mL PMA, and harvested at increasing intervals thereafter. The zf9 mRNA levels (panel C) and Zf9 protein levels (panel D) were analyzed by Northern blotting and Western blotting, respectively. Lanes 1-5, without PMA; lanes 6-10, with PMA. Each experiment was repeated 3 times with similar results and representative results are shown. (E) Superinduction of Zf9 mRNA in the presence of cycloheximide. After a preincubation in serum-free medium for 5 hours, confluent BAEC cultures were incubated for 3 hours with either fresh serum-free medium or 10% serum-containing medium in the absence or presence of 6 μg/mL cycloheximide (CHX). Cell lysates were prepared and the changes of zf9 mRNA levels were assessed by Northern blotting. Lane 1, no stimulus; lane 2, serum alone; lane 3, cycloheximide alone; lane 4, serum plus cycloheximide. The experiment was repeated 4 times with similar results and a representative result is shown.
