Stress-induced expression of Clk1. (A) Stress-induced maturation of intron 3/4–retaining Clk1 RNA. NIH-3T3 cells were incubated under heat shock (HS) condition (incubated at 43°C) for 60 min (left) or osmotic stress condition (treatment with 600 mM sorbitol) for 60 min (right). Splicing pattern of Clk1 was analyzed by RT-PCR using the same primers as in Fig. 2 A (a). The experiment was performed in triplicate. (bottom) Graphs represent the means with SEM (n = 3) of changes in the intensities after the stresses. (B) Time course analysis of heat shock–induced maturation of Clk1. NIH-3T3 cells were exposed to heat shock at 43°C for the indicated times. Splicing pattern of Clk1 was analyzed by RT-PCR using the same primers as Fig. 2 A (a and b). The maturation intermediate retaining intron 4 is indicated by arrows in a and b. (bottom) Hsp1b mRNA was used as a heat shock control, and Gapdh mRNA was used as an internal control. (C) Effect of Clk1/4 knockdown on SR protein phosphorylation. The cells were incubated with the siRNA against Clk1/4 from 3 d before the assay (lanes 4–6) with the same protocol as in Fig. S2 A. As a control, siRNA against luciferase was used (lanes 1–3). The cells were incubated at a normal temperature (37°C; lanes 1 and 4), incubated at 43°C for 1 h (lanes 2 and 5), and further incubated at 37°C for 4 h (lanes 3 and 6) as indicated in the schema. Phosphorylation states of SR proteins were estimated by immunoblotting using an antiphospho-SR protein antibody. An asterisk shows the shorter exposure for SRSF6. (bottom) The immunoblot with an antibody against α-tubulin was shown as an internal control. (D) Quantification of results in C. Phospho-SRSF4 was quantified by measuring the intensity of the top band (at ∼80 kD) of SRSF4 in C. Each bar shows the mean with SEM of three independent experiments.