Figure 8.

Sam68 nuclear bodies do not contain nascent RNAs and snRNPs. (A) HeLa cells transfected with GFP-Sam68 (a–c), GFP-Sam68ΔL1 (d–f), or GFP-PTB (g–i) were permeabilized with 50 μg/ml saponin for 5 min at 4°C and then incubated with a transcription cocktail containing Br-UTP for 20 min at 33°C. The cells were fixed, and the sites of Br-UTP incorporation were detected by immunolabeling using anti-BrdU antibody (which also recognizes Br-UTP) followed by a rhodamine-conjugated secondary antibody (b, e, and h). Colocalization was determined by confocal microscopy (c, f, and i). The arrows in panel i indicate colocalization of PNC with sites of Br-UTP incorporation. (B) HeLa cells (a–c) or HeLa cells transfected with GFP-Sam68ΔL1 (d–f) were fixed, permeabilized, and immunostained with anti-SC35 antibody followed by a secondary antibody conjugated to rhodamine (b and e). Endogenous Sam68 nuclear bodies were detected by immunolabeling using anti-Sam68 AD1 antibody followed by FITC-conjugated goat anti-rabbit antibody (a). Colocalization of SNBs and SC35 was analyzed by confocal microscopy (c and f). The arrows in panel f indicate partial overlap of Sam68ΔL1 SNBs with SC35, most likely random overlapping.