Fig 2: Immunolabeling patterns of native NUP62 and NUP214 antibodies are consistent with those produced by V5 epitope tag antibodies detecting ectopically expressed nucleoporins.Immunofluorescent images represent 3D deconvolved projections of 250 nm optical sections through 10 to 15 um of the z axis. Projections are shown on x,y, x,z, and z,y planes, as indicated. Bar represents 10 um. A. TOV112D cells were transfected with an expression plasmid for V5 epitope-tagged NUP62 and cultured for 72 hours. The distribution of NUP62 was analyzed in pairs of post-mitotic cells expressing V5 epitope-tagged NUP62. Immunofluorescence analyses of NUP62 (green) and V5 epitope-tagged NUP62 (red) were performed with Ab1 and V5 epitope tag antibodies, respectively. Blue, DAPI stain; yellow, overlap between green and red. B. TOV112D cells were transfected with an expression plasmid for V5 epitope-tagged NUP214 and cultured for 72 hours. The distribution of NUP214 was analyzed in pairs of post-mitotic cells expressing V5 epitope-tagged NUP214. The images of the pair of post-mitotic cells shown have been cropped individually to avoid interference from surrounding cells, particularly on the x,z and z,y planes. Immunofluorescence analyses of NUP214 (red) and V5 epitope-tagged NUP214 (green) were performed with Ab4 and V5 epitope tag antibodies, respectively. Blue, DAPI stain; yellow, overlap between green and red. C. TOV112D cells were transfected with an expression plasmid for V5 epitope-tagged NUP214 and cultured for 72 hours. The distributions of NUP62 and NUP214 were analyzed in pairs of post-mitotic cells expressing V5 epitope-tagged NUP214. Immunofluorescence analyses of NUP62 (green) and V5 epitope-tagged NUP214 (red) were performed with Ab1 and V5 epitope tag antibodies, respectively. Blue, DAPI stain; yellow, overlap between green and red.

Fig 3: Effects of transfection reagents and cell density on nucleoporin distribution.A. TOV112D cells were treated in the absence of exogenous DNA with Lipo293D (SignaGen Laboratories), Lipofectamine (Invitrogen), or FuGENE HD (Promega), according to the instructions provided by the manufacturers. The cells were processed for immunofluorescence using NUP62 (green) and NUP214 (red) antibodies 48 hours after treatment. Immunofluorescent images represent 3D deconvolved projections of 250 nm optical sections through 10 to 15 um of the z axis. Projections are shown on x,y, x,z, and z,y planes, as indicated. Bar represents 5 um. Blue, DAPI stain; yellow, overlap between green and red. B. TOV112D were cultured at confluence for three days, and analyzed by immunofluorescence microscopy as described above. C. TOV21G cells were cultured at confluence for three days, and analyzed by immunofluorescence microscopy as described above. D. TOV21G cell cultured at low density and analyzed by immunofluorescence microscopy as described above.

Fig 4: Detection of NUP62 and NUP214 in nuclear and cytoplasmic compartments of cultured cells.Whole cell, cytosolic, and nuclear fractions from Hs 832(C).T (Hs), TOV112D, HEK293 (293), S12, and COS7 cells were analyzed by immunoblot with antibodies to NUP62 and NUP214 (antibodies used were also used for immunofluorescence). Two T75 plates of cells at 80% confluence were used for each preparation (this does not yield equal numbers of cells). One third of the sample was taken for the whole cell extract; the remaining sample was fractionated into cytosol and nuclear components. Extracts were loaded to represent equivalent fractions of starting material. Indicated as Mr∼35K is an additional band observed in the NUP214 immunoblot. The Mr∼70K band present in the Hs832(C).(Hs) whole cell lysate fractionated with the cytosol. The Western blot analyses of the whole cell lysates were completed on single membranes for all five cell lines; however, the exposure times for Hs832(C).(Hs) and Cos 7 cells using the NUP214 antibody and for Hs832(C).(Hs) using the NUP62 antibody were longer. Consequently, those lanes are separated.

Fig 5: Total accumulation and distribution of NUP62 and NUP214 in different cell lines.Top left corner: immunoblot analyses of Hs 832(C).T (Hs), TOV112D (112D), HEK293 (293), S12, and COS7 (cos7) cell lysates. Antibodies are explained in the text. Immunofluorescent images for the indicated cell lines represent 3D deconvolved projections of 250 nm optical sections through 10 to 15 um of the z axis. Projections are shown on x,y, x,z, and z,y planes, as indicated. immunofluorescence analyses of NUP62 (green) and NUP214 (red) were generated using Ab1 and Ab4, respectively. Blue, DAPI stain; yellow, overlap between green and red. Bars represent 2 um.