Ab171074

For all immunostainings, primary antibodies (Abcam) used were rabbit anti-Nup358 at 1:200 (ab64276); rabbit anti-Nup214 at 1:200 (ab70497); mouse anti-Nup62 at 1:300 (ab96134); rabbit anti-Nup153 at 1:300 (ab171074); rabbit anti-TPR at 1:100 (ab170940); rabbit anti-lamin A at 1:200 (ab26300). Secondary antibodies: the secondary antibodies used were anti-mouse Cy3 (ab97035), anti-rabbit Cy3 (ab6939; Abcam), Alexa Fluor 647 goat anti-mouse (A21235), and Alexa Fluor 594 goat anti-rabbit (A11072; Molecular Probes).

Cells were lysed and total protein was extracted with RIPA lysis buffer (Beyotime Technology). The total protein concentration was determined with a BCA kit (Thermo Fisher). The protein samples were boiled at 95°C for 5 min, separated by 10% SDS-PAGE, and transferred to 0.45 μm PVDF membranes (Millipore). After incubation with primary antibodies overnight at 4°C, these membranes were washed with TBST buffer and incubated with secondary antibodies at room temperature. The primary antibodies were SMARCC1 (ab172638, 1:1,000, Abcam, United Kingdom), KPNA2 (ab70160, 1:10,000, Abcam, United Kingdom), NUP50 (ab151567, 1:2000, Abcam, United Kingdom), NUP153 (ab171074, 1:1,000, Abcam, United Kingdom) and β-tubulin (ab6046, 1:5000, Abcam, United Kingdom). The secondary antibodies were anti-rabbit IgG (7074,1:2000, Cell Signaling Technology, United States) and anti-mouse IgG (7076, 1:2000, Cell Signaling Technology, United States) HRP linked antibody. Immunoreactive bands were visualized by enhanced chemiluminescence using HRP Substrate Peroxide (Millipore).