Delay of SR protein rephosphorylation after heat shock by pretreatment with an SF3b inhibitor. (A) Effect of the SF3b inhibitor FR901464 on Clk1/4 RNAs. The effect of FR901464 treatment on NIH-3T3 cells (20 nM for 30 min) was analyzed by RT-PCR using primer sets of exons 2–5 (Clk1; same as shown in Fig. 2 A [a]) or exons 1–6 (Clk4) as illustrated above. Gapdh was used as an internal control. (B) Time course analysis of phosphorylation states of SR proteins during and after heat shock (HS). Recovery of the phosphorylation states were compared between NIH-3T3 cells of control (no drug-treated cells; lanes 1–5) and FR901464 pretreated (cells transiently pretreated with 20 nM FR901464 for 30 min; lanes 6–10) by immunoblotting using the antiphospho-SR protein antibody. The cells were incubated at a normal temperature (37°C; lanes 1 and 6), incubated at 43°C for 1 h (lanes 2 and 7), and further incubated at 37°C for 1 h (lanes 3 and 8), 2 h (lanes 4 and 9), and 4 h (lanes 5 and 10) as indicated in the schema. An asterisk shows the shorter exposure for SRSF6. Clk1/4 was detected using a Clk1 antibody which recognizes Clk1 and Clk4. (bottom) The immunoblot with an antibody against α-tubulin was shown as an internal control. (C) Quantification of the results in B. Gray bars and closed bars indicate the control and the FR901464-pretreated cells, respectively. Phospho-SRSF4 was quantified by measuring the intensity of the top band (at ∼80 kD) of SRSF4 in B. Each bar shows the mean with SEM of three independent experiments. Asterisks indicate a significant difference (*, P < 0.01; **, P < 0.05; each actual p-value is also indicated).