Figure 2. Nuclear localization of the... | Rockefeller University Press

Figure 2.

$Figure 2. Nuclear localization of the Clk1 intron-retaining RNA. (A) RT-PCR analysis of nucleus/cytoplasm subfraction. Positions of the primers used in a, b, and c and the hybridization probe used in C are indicated in the schema by arrows and a bar, respectively. The same primer pairs were used in the PCR analyses of Fig. 3, Fig. 5, Fig. 6, and Fig. 7. Intron 3/4–retaining Clk1 RNA (a band at 1,804 bp in a and bands shown in b and c) and Neat1 RNA were enriched in the nuclear fraction, whereas mature Clk1 (546 bp in a) and Gapdh mRNAs were enriched in the cytoplasm. (B) The ratio of nuclear to cytosolic mRNAs quantitated by real-time PCR. Each bar shows the mean with SEM of three independent experiments. (C) In situ hybridization of HeLa cells using a human Clk1 intron 4 probe. Nuclear signals of the Clk1 intron 3/4–retaining RNA, visualized by the antisense probe, were detected in control cells and disappeared in the cells fixed after 50 µM TG003 treatment for 30 min. Nuclei were stained with Hoechst 33342. Bar, 20 µm.$

Nuclear localization of the Clk1 intron-retaining RNA. (A) RT-PCR analysis of nucleus/cytoplasm subfraction. Positions of the primers used in a, b, and c and the hybridization probe used in C are indicated in the schema by arrows and a bar, respectively. The same primer pairs were used in the PCR analyses of Fig. 3, Fig. 5, Fig. 6, and Fig. 7. Intron 3/4–retaining Clk1 RNA (a band at 1,804 bp in a and bands shown in b and c) and Neat1 RNA were enriched in the nuclear fraction, whereas mature Clk1 (546 bp in a) and Gapdh mRNAs were enriched in the cytoplasm. (B) The ratio of nuclear to cytosolic mRNAs quantitated by real-time PCR. Each bar shows the mean with SEM of three independent experiments. (C) In situ hybridization of HeLa cells using a human Clk1 intron 4 probe. Nuclear signals of the Clk1 intron 3/4–retaining RNA, visualized by the antisense probe, were detected in control cells and disappeared in the cells fixed after 50 µM TG003 treatment for 30 min. Nuclei were stained with Hoechst 33342. Bar, 20 µm.

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