U1-70K knockdown results in co-depletion of U1C protein and in U1-70K/U1C double-deficient U1 snRNPs.

<p>(<b>A–B</b>) 96 hours after siRNA transfection, whole-cell lysates were prepared from control- (ctr) and U1-70K-knockdown- (Δ70K) cells. (<b>A</b>) U1-70K knockdown in HeLa cells results in co-depletion of U1C. Whole-cell lysates were analyzed by SDS-PAGE and Western blot, detecting U1-70K, γ-tubulin, and U1C. (<b>B</b>) U1-70K/U1C double-deficient U1 snRNPs in U1-70K-knockdown HeLa cells. U1 snRNPs were affinity-purified from whole-cell lysates (5% of input, lanes 1 and 3; 2/3 of purified material, lanes 2 and 4) and analyzed by SDS-PAGE and Western blot, detecting U1-70K, U1A, and U1C. U1 snRNA was detected by Northern blotting, analyzing 1/3 of the purified material. Asterisks mark unspecific bands detected by the anti-U1-70K antibody. (<b>C</b>) Exon 7a skipping upon U1-70K knockdown. 72 hours after siRNA transfection, the U1-70K wildtype minigene construct (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003856#pgen-1003856-g004" target="_blank"><b>Figure 4A</b></a>) was transfected into HeLa cells, and 24 hours later minigene splicing patterns were analyzed by RT-PCR, comparing control- (ctr) and U1-70K- (Δ70K) knockdown samples. Specific primer sets were used to detect alternative 3′ splice site activation (product 7-7a), exon 7a inclusion (product 7-7a-8; letters indicate which cryptic 5′ splice sites are used, with parentheses marking the less frequently used splice site; see <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003856#pgen.1003856.s003" target="_blank">Figure S3A</a></b>), exon 7a skipping (product 7–8), and as a loading control, exon 7 alone (product 7). The identities of the splicing products are depicted on the right. (<b>D</b>) U1C mRNA is stable after U1-70K knockdown. After four days of U1-70K knockdown in HeLa cells, total RNA was isolated from control- (ctr) and U1-70K- (Δ70K) knockdown cells. Endogenous mRNA levels of U1-70K, U1C, and β-actin (as indicated on the right) were analyzed by RT-PCR. The numbers given in parentheses refer to the amplified exons. M, DNA size markers (in bp).