Anti sorbs2

Heart tissues were homogenized in radioimmunoprecipitation assay buffer with 0.5‐mm steel beads at speed 8 for 4 minutes, followed by speed 10 for 1 minute using a Bullet Blender (Next Advance). Lysate samples were denatured at 95°C for 10 minutes and loaded onto an SDS‐PAGE and Western blotting. Anti‐Sorbs2 (Sigma, catalog No. SAB4200183), anti–connexin 43 (Cx43) (Cell Signaling, catalog No. 3512), anti–α‐catenin (Thermofisher Scientific, catalog No. 71‐1200), anti–β‐catenin (Thermofisher Scientific, catalog No. 13‐8400), anti–desmoglein 1/2 (Progen, catalog No. 61002), anti‐plakoglobin (Progen, catalog No. 65105), anti–N‐cadherin (Sigma, catalog No. C1821), and anti‐GAPDH (Santa Cruz Biotechnology, catalog No. sc‐25778) antibodies were used.

The heart samples harvested from mice were embedded in tissue freezing medium and stored at −80°C, followed by sectioning at 10 μm using a cryostat (Leica CM3050 S). The slides were subjected to immunostaining using a previously described protocol.³⁴ The following antibodies were used: anti‐Sorbs2 (Sigma, catalog No. SAB4200183) at 1:200, anti‐Cx43 (Cell Signaling, catalog No. 3512) at 1:200, anti–N‐cadherin (Sigma, catalog No. C1821) and anti–β‐catenin (Thermo Fisher Scientific, catalog No. 13‐8400) at 1:200, and anti‐plakoglobin (Progen, catalog No. 65105) at 1:200. A Zeiss LSM 780 confocal microscope was used to acquire the image. The tile scan function was used to acquire images covering a complete mouse heart. For each group, 5 to 10 slides were imaged and analyzed. The distribution of fluorescence density was analyzed by pseudoline scan using Zen software (Zeiss). The weighted colocalization of Sorbs2 with other ICD proteins was determined using a colocalization function in Zen software.