Ab106393

Cardiomyocytes were divided into four groups, lysates of cardiomyocytes were prepared in RIPA buffer, and protein concentration was determined by BCA protein assay (Thermo Fisher Scientific, US). Equal amounts (30 μg) of proteins were separated on 10% SDS-PAGE (100 V for 120 min) and transferred into nitrocellulose membranes (120 min at 100 V) using standard procedures. The binding of nonspecific proteins to the membrane was blocked with blocking buffer (5% nonfat milk) at room temperature for 2 h. The membranes were probed with anti-CaMKII (ab52476, Abcam), anti-RIPK1 (ab106393, Abcam), anti-RIPK3 (sc-374639, Santa Cruz), and anti-β-actin (T40104, abmart) at 4°C overnight. After the membrane was washed three times with TBST (20 mM Tris, 500 mM NaCl, and 0.1% Tween 20) for 10 min each time at room temperature, the membrane was incubated with the second antibody (1 : 20000) for 1 h at 37°C. Then, the membrane was washed three times with TBST for 10 min each time at room temperature. Detection was performed using an ECL chemiluminescence kit (PK10003, Proteintech). Finally, the PVDF membrane was placed in the Bio-Rad ChemiDocXRS gel imaging system for photography under exposure, and band intensities were quantified using Image Lab software. Three independent replicates were conducted for this experiment.

Mouse monoclonal antibodies against AQP1 (ab9566) and RIPK1 (ab72139) were purchased from Abcam (Cambridge, MA). Mouse monoclonal antibodies against RIPK1 (610458), caspase-8 (9746), and RIPK3 (sc-374639) were bought from BD Biosciences (San Jose, CA), Cell Signaling Technology (Danvers, MA), and Santa Cruz Biotechnology (Dallas, TX), respectively. The rabbit monoclonal antibody against p-MLKL (S345) (ab196436), p-RIPK3 (S227) (ab209384) and p-RIPK3 (S232) (ab195117), and rabbit polyclonal antibody against RIPK1 (ab106393) and MLKL (ab194699) were purchased from Abcam. The rabbit polyclonal antibody against cleaved caspase-3 (9661) and caspase-3 (9662) were bought from Cell Signaling Technology. The rabbit monoclonal anti-β-actin antibody (AC026) and anti-α-tubulin antibody (AC013) were from Abclonal (Wuhan, China). The cell transfection regent (F231-02) was purchased from TransGen Biotech (Beijing, China). The point mutation plasmids of RIPK1 were constructed using the HiFi HotStart DNA Polymerase (KAPA Biosystems, Roche, Switzerland).