Anti emerin

For Western blotting, cells were washed in PBS, scraped in SDS gel-loading buffer, and boiled for 5 min. Proteins of total extracts were separated by SDS-PAGE and transferred to nitrocellulose membranes. Monoclonal antibody against HA was purchased from BioLegend. Anti-HA goat was from Bethyl Laboratories. Anti-emerin and anti-lamin A/C antibodies were from Abcam and Cell Signaling. Anti-tubulin, anti-gH2AX, and anti-ERK were from Cell Signaling. Anti-GAPDH, anti-actin, anti-lamin B, anti-RanGAP, anti-RanBP, and anti-phospho-ERK antibodies were from Santa Cruz Biotechnology. Anti-VP24 was from Biorbyt. Anti-VP35 and anti-NP were from Genetex. Anti-BAF antibody was from Abcam.

Proteins from self-beating embryoid bodies (EBs) were extracted with RIPA buffer (Nacalai Tesque, Japan). The proteins were loaded on Mini-PROTEAN TGX Gels (Bio-Rad, 4% to 15%) and then electrophoretically separated and transferred onto nitrocellulose blotting membranes (GE Healthcare). The primary antibodies were anti-emerin (Abcam), anti-SYNE1 (Millipore), and anti-GAPDH (Cell Signaling Technology). Protein expression was visualized with appropriate horseradish peroxidase-conjugated secondary antibodies (anti-mouse HRP 1:2000, anti-rabbit HRP 1:2000) and enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA), and was detected using an LAS-3000 luminoimager (Fujifilm Techno Products, Ayase-shi, Kanagawa, Japan). Protein bands were quantified using the ImageJ software (NIH, Bethesda, MD, USA).