Ab26300

Previously fixed coverslips were subjected to two washes with 1× PBS (5 min each). Blocking was performed for 30 min using 1 % bovine serum albumin (BSA) in 1× PBS at RT. Primary antibodies used were as follows: Mouse anti-Lamin B2 (Abcam (ab8983), 1:600) and Rabbit anti-Lamin A (Abcam (ab26300), 1:500). Antibody dilutions were prepared in 0.5 % BSA in 1× PBS and incubated for 90 min at RT. Secondary antibodies used were goat anti-mouse IgG–Alexa 633 (Invitrogen (A-21052) 1:1000) and goat anti-rabbit IgG–Alexa 488 (Invitrogen (A-11034) 1:1000) for 1 h at RT. Post fixation and post permeabilization were subsequently performed with 4 % PFA (5 min RT) and 0.5 % Triton X-100 in 1× PBS (5 min RT), respectively. This was followed by two washes each with 1× PBS (5 min each) and 50 % FA/2× SSC (pH 7.4). The probe for ZNF570 was equilibrated at 37 °C for 5 min followed by denaturation at 80 °C for 5 min and quick chilled on ice for 2 min. Pre-annealing was performed at 37 °C for 30–40 min. This denatured probe (3–4 μL) was spotted onto the fixed cells that were subjected to immunostaining for Lamin A and Lamin B2 and subjected to co-denaturation at 80 °C for 5 min. Hybridization was for 48 h in a humidified box at 37 °C.

Western blot (WB) was carried out as our previous method.⁴⁴ (link), ⁴⁵ (link), ⁵⁵ (link) The following primary antibodies were applied: ^Ser536p‐NF‐κB p65 (Cat. No.: #3033), NF‐κB p65 (Cat. No.: #8242), ^Ser473p‐Akt (Cat. No.: #4060), Akt (Cat. No.: #9272), Bax (Cat. No.: #2772), cleaved caspase‐3 (Cat. No.: #9664) (1:1000, CST, Danvers, MA, USA), Bcl‐2 (Cat. No.: ab59348, 1:500, Abcam), ^Tyr607p‐PI3K (Cat. No.: ab182651, 1:1000, Abcam), and PI3K (Cat. No.: BSM‐33219 M, 1:1000, Bioss, Beijing, China). β‐actin (Cat. No.: ab8227, 1:1000, Abcam) and Lamin A (Cat. No.: ab26300, 1:3000, Abcam) were employed as the internal reference. WB protein bands were quantified by ImageJ software (National Institutes of Health, Baltimore, MD, USA). Protein expression levels were indicated by the ratio of interest protein bands to that of β‐actin or Lamin A bands.

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).