Emerin

For immunofluorescent staining, isolated adult cardiomyocytes were fixed for 30 min, room temperature with 2%PFA/PBS/0.1%Triton x-100. Samples were then stained with antibodies for Lamin A/C (A gift from Larry Gerace), Emerin (Santa Cruz), Sun2 (Sigma), α-actinin (Sigma) or Nesprin 2(Abgent). Samples were then stained with secondary antibodies conjugated to CY3 (Jackson ImmunoResearch Inc.). Samples were then incubated with DAPI (Invitrogen) and Phalloidin Alexa-488 (Invitrogen). Samples then imaged using standard confocal microscopy (Olympus FV1000 Confocal). Image analyses were then performed and samples quantified using either Velocity high performance 3D imaging software (PerkinElmer) or ImagePro3D software analyses (MediaCybernetics).

Antibodies used in this study are: LMO7 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-FAK (Cell Signaling, Danvers, MA, USA), Emerin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-catenin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), NKCC2 Cell Signaling, Danvers, MA, USA), E-cadherin (Arigo, HsinChu, Taiwan), β-actin (Arigo, HsinChu, Taiwan), ZO-1 (Thermo Fisher Scientific, Waltham, MA, USA), and p-Paxillin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Specimens were imaged with DM16000B epifluorescence microscopy (Leica, Wetzlar, Germany). Images were analyzed with Software Image-Pro Plus 8.0 (Media Cybernetics, Rockville, MD, USA). Detailed material information is listed in the key resource table (Supplementary Table S3).

The sources of the antibodies to the different markers were as follows: emerin (mouse mAb IgG2b, used at 1/250 for immunofluorescence analysis (IF) and at 1/500 for western blot (WB); sc-25284); PML (mouse mAb IgG1; used at 1/250 for IF and 1/500 for WB; sc-377390); lamin A/C (mouse mAb; used at 1/250 for IF and WB; sc-7292) were obtained from Santa Cruz Biotechnology, Inc; GAPDH (mouse mAb IgG1; used at 1/20,000 for WB; AM4300) was from ThermoFisher Sci.; α-tubulin (mouse mAb IgG1; used at 1/500 for IF and 1/200 for WB; T6074) was from Sigma-Aldrich; caveolin-1 (mouse mAb IgG1, used at 1/2,000 for WB; 610,058) was from BD Biosci. The hybridoma producing antibodies to SC35 (mouse mAb IgG1, used at 1/50 for IF; CRL-2031) and myc (9E10) (mouse mAb IgG1, used at 1/500 for IF and WB; CRL-1729) were obtained from the American Type Culture Collection (ATCC). Secondary antibodies conjugated to Alexa Fluor-488, -555, -594 and -647 were from ThermoFisher Sci. 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI; 268,298) was from Merck, and SiR-DNA (CY-SC007) from Cytoskeleton Inc. Horseradish peroxidase-labeled secondary antibodies were from Jackson Immunoresearch Laboratories, Inc.