Figure 1.

RNA interference-mediated depletion of TIA proteins in HeLa cells. (a) Immunoblot analysis of HeLa cell lysates (5 μg (lane 1; C(1/5)) or 25 μg (lanes 2 to 5)) prepared 72 h after transfection with siRNAs against control (C; lanes 1 and 2), TIA-1 (lane 3), TIAR (lane 4), and TIA-1 plus TIAR (lane 5; 1+R). TIA-1(a/b) and TIAR(a/b) refer to the major isoforms of TIA-1 and TIAR, respectively. The blot was probed with antibodies against TIA-1, TIAR, U2AF65 and α-tubulin proteins, as indicated. Molecular weight markers and the identities of protein bands are shown. The intensities of the different protein bands from immunoblots were quantified by densitometry. The represented values were normalized and are expressed relative to α-tubulin (α-Tub), whose value is fixed arbitrarily to 1, and are means ± standard error of the mean (SEM; n = 10; *P < 0.05). (b) Cytoplasmic mRNAs from post-transfected HeLa cells in (a) were analyzed by RT-PCR. Positions of size markers and the predicted alternatively spliced products are indicated. Quantification of relative levels of TIA-1, TIAR and β-actin mRNAs in the above post-tranfected HeLa cells by real time RT-PCR. The represented values were normalized and are expressed relative to β-actin mRNA, whose value is fixed arbitrarily to 1, and are means ± SEM (n = 3; *P < 0.01).