FIG. 3.

TAP-3xUBA and TAP-4xUBA localize to the nuclear rim. (A) HeLa cells were transfected with a plasmid expressing GFP-TAP. When cells were directly fixed in formaldehyde, TAP was predominantly detected throughout the nucleoplasm, excluding nucleoli (−Triton). When cells were extracted with Triton X-100 prior to fixation, a punctate labeling pattern was visible at the nuclear periphery (+Triton). (B to F) HeLa cells were transfected with plasmids expressing GFP fusions of TAP derivatives as indicated. Approximately 20 h after transfection, cells were fixed in formaldehyde and directly observed with a confocal microscope. TAP-2xNTF2, TAP-1xUBA, and TAP-2xUBA were detected throughout the nucleoplasm (B, C, and D). An additional punctate labeling pattern was visible at the nuclear rim for TAP-2xUBA. TAP-3xUBA and TAP-4xUBA localized predominantly at the nuclear rim (E, F). (G to R) Human 293 cells were transfected with plasmids encoding GFP fusions of TAP derivatives as indicated. Approximately 20 h after transfection, bulk poly(A)⁺ RNA was detected by FISH with an indocarbocyanine-labeled oligo(dT) probe. All images were taken with the same settings so the GFP signals could be compared directly. In panels K, M, O, and Q, examples of cells expressing TAP-3xUBA or TAP-4xUBA at different levels are shown. In about 10 to 20% of transfected cells, TAP-3xUBA and TAP-4xUBA were expressed at higher levels (M and Q), resulting in a strong accumulation of the poly(A)⁺ signal within the nucleus (N and R). wt, wild type.