H9c2 cells were seeded at glass coverslips placed in a 24-well dish. After being treated according to the different group protocols, the coverslips were washed with cold phosphate-buffered saline (PBS) and placed in 4% paraformaldehyde for 20 minutes at room temperature. After being permeabilized with 0.1% Triton X-100, the cells were blocked with goat serum for 1 hour at room temperature. The process described above is similar to our previous study [21 (link)]. Next, the cells were incubated with primary rabbit anti-phospho-JNK (1 : 50) and mouse anti-Sab (1 : 50) antibodies overnight at 4°C. Sab was detected using an Alexa Fluor™ 594-conjugated anti-mouse secondary antibody (Thermo-Fisher Scientific, Waltham, MA, USA), while phospho-JNK was detected using an Alexa Fluor™ 488-conjugated anti-rabbit antibody (Thermo-Fisher Scientific, Waltham, MA, USA). Finally, the cover glasses were mounted on a glass slide, and images were observed with a confocal laser scanning microscope (Carl Zeiss AG, Oberkochen, Germany).
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