Ez gel imager

The total protein content from myocardial tissue or cells was extracted for measurement of protein concentration using the bicinchoninic acid assay (ThermoFisher). The extracted protein was added into the loading buffer and boiled at 95 °C for 10 min. Next, the protein sample (30 μg) was separated by 10% (w/v) electrophpresis and transferred onto polyvinylidene difluoride membranes. The membranes were blocked using 5% BSA for 1 h and cultured with the primary antibodies at 4 °C overnight. After being washed by tris buffered saline tween (TBST), the membranes were incubated with secondary antibody for 1 h and then washed by TBST before enhanced chemiluminescence developing and visualization using Bio-Rad GelEZ imager (Bio-Rad, Inc., Hercules, CA, USA). Image J software (National Institutes of Health, Bethesda, Maryland, USA) was utilized to analyze the gray value of target bands. The antibodies used were IP3R1 (1:2000, ab264281, Abcam), IgG (1:2000, ab205718, Abcam), NLRP3 (1:1000, ab263899, Abcam), ASC (1:1000, ab175449, Abcam), GSDMD (1:2000, ab219800, Abcam), GAPDH (1:1000, ab8245, Abcam), and ERP44 (1:1000, #3798, Cell Signaling Technology, Danvers, MA, USA).

Total nucleic acids were extracted using the modified CTAB procedure described by Abarshi et al. [16 (link)]. Approximately 100 mg of leaf material was grounded in 1 mL CTAB buffer and 600 μL of the extract was transferred into a 1.5-ml tube and incubated at 60 °C for 10 min. Equal volume of phenol:chloroform: isoamyl alcohol (25:24:1) was added to the extract and then centrifuged at 12,000 g for 10 min. The supernatant was transferred to a new 1.5 ml tube, to which 300 μL of ice-cold iso-propanol was added and incubated at −20 °C for 60 min and then tubes were centrifuged at 12,000 g for 10 min to precipitate nucleic acids. The precipitated pellet was washed with 0.5 mL of 70% (v/v) ethanol, centrifuged for 5 min, and the pellet was air-dried at room temperature. The final pellet was suspended in 50 μL of sterile distilled water and stored at −20 °C until used. Total nucleic acid was quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific, UK) and nucleic acid integrity was analyzed by electrophoresing a 4 μL aliquot on a Tris-Acetate EDTA (TAE) agarose gel (Sigma, France) at 110 V for one h. Gels were stained with 0.5 μg/mL ethidium bromide and visualized under ultraviolet (UV 302 nm) light using a EZ Gel Imager (Bio-Rad, France).