A19524

For immunostaining, cells were washed with PBS three times and then fixed in 4% paraformaldehyde. Cells were frozen in liquid nitrogen and fixed on slides. Slides were blocked in PBSTA [PBS with 0.1% Triton X-100 and 3% bovine albumin (BSA)] for 1 h at room temperature; with primary antibodies, overnight at 4°C; and with secondary antibodies, for 1 h at room temperature. Cell nuclei were stained by using Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; H-1800–2, Vector Laboratories). Anti-lamin A/C [rabbit monoclonal antibody (mAb), A19524] was purchased from ABclonal Technology (WuHan, China). Anti-GFP (mouse mAb, ab1218) and anti-fibrillarin (mouse mAb, ab4566) were purchased from Abcam Technology. Immunofluorescence images were collected by using a Zeiss LSM 980 Upright laser scanning confocal microscope with a ×63 objective. Live-cell images were collected by using a Nikon ECLIPSE Ti-E inverted microscope with either ×40 or ×20 objectives.

The 293 cells were collected and lysed in 1× loading buffer (9156, Takara). After sonication, lysates were centrifuged at 14 000 × g for 30 min. Total proteins were resolved with 7.5% TGX polyacrylamide gels (1610171, Bio-Rad) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore) by reverse electrophoresis. After being blocked, membranes were stained with anti-GFP (1:2000 dilution; ab1218, Abcam), anti-lamin A/C (1:10000 dilution; A19524, ABclonal) or anti-GAPDH (1:2000 dilution; 2118, Cell Signaling Technology) antibodies. Chemiluminescence signals were collected using Amersham Imager 800 (GE).