Ab3447

The placenta tissues were taken from the liquid nitrogen tank and homogenized by grinding the tissue in liquid nitrogen. The homogenate was collected, added to the tissue lysis buffer (P0013, Beyotime) with PMSF and cocktail, and sonicated (10s, 30s, 5-10 cycles) to make the tissue fully lysed. The supernatant was collected by centrifugation (12000 rpm/min, 10-15 min) and set aside at -80°C. The proteins were boiled with 5×SDS loading buffer, resolved by SDS-PAGE, and measured by indicated antibodies and anti-rabbit or anti-mouse secondary antibody conjugated with horseradish peroxidase. Specific bands were visualized by enhanced chemiluminescence (ECL). Antibodies against the following epitopes or proteins were obtained from the indicated suppliers: NFATC4 (ab3447, Abcam), BRAF (20899-1-AP, Proteintech) and GAPDH (60004-1-Ig, Proteintech).

The frozen myocardial tissue from Days 1, 7, 14, and 28 after radiation was weighed at 20 mg, added to 200 μL of tissue lysis solution (1:100 with protease inhibitor), homogenized in 60 Hz 10 times (45 s each time, interval 15 s), and centrifugated at 12,000 r in 4 °C for 15 min. The supernatant was collected as total protein solution. The concentration of protein solution was measured using the BCA method, and the loading buffer and protein solution (4:1) were mixed and then denatured at 95 °C for 10 min. After the gel preparation, electrophoresis, membrane transfer, and closure, the anti-JNK (Rabbit, Abcam, ab179461, Cambridge, UK), anti-p-JNK (Rabbit, Abcam, ab219584, Cambridge, UK), anti-NFATc4 (Rabbit, Abcam, ab3447, Cambridge, UK), anti-HSF1(Rabbit, Abcam, ab52757, Cambridge, UK) (diluted to 1:1000 with TBST), and anti-GAPDH (Rabbit, Abcam, ab9485, Cambridge, UK) (diluted to 1:10,000 with TBST) were added to the PVDF membranes and incubated overnight at 4 °C with slow shaking. The next day, the PVDF membranes were washed 3 × 10 min with TBST, added to HRP-labeled secondary antibody IgG (Abclonal, Boston, USA) (diluted to 1:10,000 with TBST), shaken slowly at room temperature for 1 h, and washed with TBST for 3 × 10 min. The targeted proteins were detected by enhanced chemiluminescence (ECL), calculated by Image J.

Antibodies against the following proteins were used for immunohistochemistry: RyR2 (C3–33; Sigma-Aldrich, St. Louis, MO, USA), CaM (EP799Y, Abcam, Cambridge, UK), HDAC5 ((NBP2-22152, Novus Biologicals), NFATc4 (ab3447, Abcam, Cambridge, UK).