Serca2a

Western analysis was performed on flash frozen LV samples as previously described44 (link)45 (link) using the following primary antibodies: SERCA2a (Thermo Scientific), NCX1 (Thermo scientific), collagen-I and collagen-III (Abcam), sirtuin-1 and FOXO1 (Cell signaling Inc), total and phospho (threonine-172) AMPK (Cell signaling Inc), and subsequently incubated with HRP conjugated secondary antibodies31 (link). Immunoprecipitation was performed with slight modification as described previously31 (link). Total protein lysate (100–200 μg) from flash frozen LV were incubated with 5 μg of anti-acetyl-Lysine (Millipore). The immune complex was captured by adding 50 μl protein A/G Plus-agarose beads (Sc-2003) with gentle rocking for 6 hr at 4 °C; the tubes were then centrifuged at 12000 g for 3 min and the supernatants were discarded. The pellets were gently washed with ice cold PBS and the immune complex resuspended in 60 μl of 2X Laemmli sample buffer and resolved on 8% SDS-PAGE then transferred to Immobilion PVDF membranes (Millipore) using a Trans-blot cell (Bio-Rad laboratories, Hercules CA, USA). Membranes were stained for total amount of protein as a loading control using MemCode (Thermo Scientific).

Changes in the expression of ion channels and Ca²⁺-modulated proteins were investigated through Western blotting. RVOT tissues were homogenized and centrifuged in buffer systems. Equal amounts of total protein were separated through 5% or 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The resultant protein bands were electrophoretically transferred onto polyvinylidene difluoride membranes. For the immunofluorescence-based detection of gap junction proteins, all blots were stained with primary antibodies against sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase 2a (SERCA2a), Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) phosphorylated at Thr286 (pCaMKII), RyR2, total phospholamban (Thermo Fisher Scientific, Rockford, IL, USA), phosphorylated RyR at Ser2808, phospholamban phosphorylated at Thr17 (Badrilla, UK), catalytic subunit of protein kinase A (BD BioSciences, USA), Cav1.2, NLRP3, nuclear factor (NF)-κB, IL-1β, and glyceraldehyde-3-phosphate dehydrogenase (MBL, Japan); all secondary antibodies were conjugated with horseradish peroxidase. Bound antibodies were detected using an enhanced chemiluminescence detection system and were analyzed using the AlphaEaseFC software. To ensure equal protein loading, all target bands were normalized to a band of glyceraldehyde-3-phosphate dehydrogenase.