FIGURE 7:

P90 RSK facilitates Chk1 phosphorylation not only at Ser-280 but also at Ser-296 by Chk1 and at Ser-345 by ATR after UV irradiation. (A–C) Serum starvation, agent treatment, and serum stimulation were performed as described in Materials and Methods. At 10 min after serum addition, cells were irradiated with UV and then incubated at an indicated time (B) or 30 min (C). Experimental procedures are summarized in a schema (A). Scale bar, 10 μm (C). (D) Each Tet-On RPE1 cell line was transfected with Chk1 3′UTR siRNA according to the reverse transfection procedures (Invitrogen). At 4 h after transfection, the medium was replaced with the fresh growing medium containing Dox. At 24 h after transfection, cells were irradiated with UV light at a dose of 20 J/m² and then incubated at an indicated time. After treatment, cells were subjected to anti-Myc immunoprecipitation (IP), followed by immunoblotting with the indicated antibody. Experimental procedures are also summarized in a schema (left).