Anti lamin b1

Formalin-fixed, paraffin-embedded tissue sections (6-µm thick) were acquired from the Department of Pathology, Keio University School of Medicine (Tokyo, Japan) and the Department of Neurology, Tohoku University Graduate School of Medicine (Sendai, Japan). This study adhered to the principles of the Declaration of Helsinki and was approved by the Ethics Committee of the Keio University School of Medicine (No. 20080016 and No. 20160273) and the Tohoku University Graduate School of Medicine (No. 20221927). Supplementary Table 1 presents patient demographics.
Tissue sections were gradually rehydrated with xylene three times, each for 10 min, 100% ethanol twice for 30 s, 90% for 30 s, 70% for 30 s, 50% for 30 s, and finally rinsed three times with dH₂O. Antigen retrieval was performed using an autoclave for 10 min at 120°C with target retrieval solution (Dako, pH = 6.0). After the slides were cooled, they were washed three times, each for 5 min, with 1 × PBS, permeabilized with 0.25% Triton X-100 diluted in 1× PBS for 10 min and washed three times, each for 5 min, in 1× PBS. Slides were incubated in 0.3% H₂O₂ for 1 h and blocked with 5% BSA at room temperature for 20 min. After blocking, slides were incubated overnight at 4°C with primary antibody diluted in 5% BSA. The primary antibodies used were as follows: anti-Lamin B1 (Abcam, #16048, 1:500) and anti-Nup62 (BD Bioscience, #610497, 1:200). After incubating with primary antibodies, slides were washed three times, each for 5 min, with 1× PBS, then incubated with the secondary antibody (biotinylated secondary antibody, 1:1000) diluted in 5% BSA at room temperature for 1 h. Slides were then washed three times, each for 5 min, in 1× PBS and stained with horseradish peroxidase/3,3′-diaminobenzidine tetrahydrochloride (DAB) using the Vectastain Elite ABC Kit (Vector Laboratories) until the slides reached the desired colour intensity. Slides were dehydrated using ethanol (50%, 70%, 90% and 100%) for 30 s each, and rinsed three times with xylene. Finally, slides were coverslipped using a mounting solution (Mount Quick). Immunohistochemical staining images were obtained using a Keyence BZ7000 microscope.

The proteins were separated on polyacrylamide gels and transferred onto a nitrocellulose membrane using the Trans-Blot Turbo Transfer System (Bio-Rad). The membranes were blocked with 5% NFDM in TBST (50 mM Tris/HCl pH 8, 150 mM NaCl, 0.1% Tween-20) and incubated with primary antibodies for 1 hr at RT. The primary antibodies used for WB were rabbit-polyclonal anti-WRNIP1 (Bethyl Laboratories; 1:2500), mouse-monoclonal anti-FLAG (Sigma-Aldrich; 1:1000), mouse-polyclonal anti-GAPDH (Millipore; 1:5000), mouse-monoclonal anti-FANCD2 (Santa Cruz Biotecnology;1:500), rabbit-polyclonal anti-RAD18 (Abcam; 1:2000) and rabbit-polyclonal anti-LAMIN B1 (Abcam; 1:30,000).
The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies specific to the respective species (Santa Cruz Biotechnology; 1:20,000) for 1 hr at RT. Visualisation of the signal was achieved using Western Bright ECL HRP substrate (Advansta) and imaged with Chemidoc XSR+ (Chemidoc Imaging Systems, Bio-Rad).