Ab219584

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.