Requirement of Clk1/4 intron retention for rapid recovery of SR protein phosphorylation. (A) mRNA levels of SR protein kinases. mRNA expressions of SR protein kinases and other genes during and after heat shock in the control or FR901464-pretreated cells (prepared by the same protocol as for Fig. 6 B) were analyzed by RT-PCR. Primers of Clk2, Hsp1b, and Clk1/4 were the same as for Fig.1 C, Fig. 5 B, and Fig. 6 A, respectively. Detailed information of primers of other mRNAs is described in Materials and methods. Clk2 mRNA was rarely detected in NIH-3T3 cells. (B) Rescue experiment with exogenous Clk1. The NIH-3T3 cells on a 35-mm dish were transfected with 1 µg pME-HA-mClk1 vector, which expresses HA-tagged mClk1, or a pME-HA empty vector 16–18 h before the time course experiment same as in Fig. 6 B. The cells were incubated at a normal temperature (37°C; lanes 1, 6, and 11), incubated at 43°C for 1 h (lanes 2, 7, and 12), and further incubated at 37°C for 1 h (lanes 3, 8, and 13), 2 h (lanes 4, 9, and 14), and 4 h (lanes 5, 10, and 15). An asterisk shows the shorter exposure for SRSF6. (bottom) The immunoblot with an antibody against α-tubulin was shown as an internal control. (C) Cancelation of the rephosphorylation by Clk1/4 inhibitor. TG003-mediated inhibition of Clk1/4 activity during the recovery phase delayed the recoveries of the SR protein phosphorylations, estimated by immunoblotting using the antiphospho-SR protein antibody of NIH-3T3 cells without the drug treatment (control; lanes 1–5), and the cells treated with 10 µM TG003 during the recovery phase (TG003; lanes 6–11). The cells were incubated at a normal temperature (37°C; lane 1), incubated at 43°C for 1 h (lane 2), and further incubated at 37°C for 1 h (lanes 3, 6, and 9), 2 h (lanes 4, 7, and 10), and 4 h (lanes 5, 8, and 11). The cells of lanes 9–11 were not exposed to heat shock. An asterisk shows the shorter exposure for SRSF6. (bottom) GAPDH was used as an internal control.