2.7 Western blotting assay
Western blotting was conducted using material extracted from the atrium,
as previously described (Feng et al., 2020; Xu et al., 2012; Xu et al.,
2010). Briefly, atrial tissue was homogenized with PRO-PREP protein
extract (iNtRON Biotechnology Inc., Kyungki-Do, Korea), and 10 µg of
protein was subjected to SDS-PAGE on a polyacrylamide gel (Bio-Rad
Laboratory, Hercules, CA, USA). Subsequently, the proteins were
transferred to the membrane using semi-dry electroblotting. The
resulting membranes were incubated overnight at 4°C with the following
primary antibodies: p-RyR2(Ser2814) (A010-31AP; Badrilla), ox-CaMKII
(07-1387; Merck), CACNA1C (ab84814; Abcam), TRPC3 (ab51560; Abcam),
TRPC6 (ab62461; Abcam), p-JNK (#4671S; Cell Signaling Technology), JNK
(#9258; Cell Signaling Technology), p-ERK (#9101S; Cell Signaling
Technology), ERK (#9102; Cell Signaling Technology), p-NF-κB p65
(Ser536) (#3033; Cell Signaling Technology), and GAPDH (#2118S; Cell
Signaling Technology). The membranes were then incubated with the
appropriate secondary antibodies, namely HRP-conjugated goat anti-rabbit
IgG (ab6721; Abcam) or HRP-conjugated rabbit anti-mouse IgG (ab97046;
Abcam), for 1 h at room temperature. Immunoreactions were detected using
enhanced chemiluminescence (ECL Prime western blotting Detection; GE
Healthcare).