Ripk3

Frozen liver tissues or cells were treated with protein extraction reagent supplemented with protease inhibitor cocktail (Bimake, B14002). Extracted protein samples were used for electrophoresis and then transferred onto polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5%(w/v) skim milk (BD Bioscience, USA) for 1 h at room temperature, and then incubated with the following primary antibodies overnight at 4 °C: TWEAK (Abcam, ab37170, 1:1000), Tnfrsf12a(Abcam, ab109365, 1:1000), Phospho-RIPK1 (Cell Signaling Technology, 31122S, 1:1000), cleaved caspase-8 (Cell Signaling Technology, 8592T, 1:1000), cleaved caspase-3 (Cell Signaling Technology, 9664S, 1:1000), cleaved PARP (Cell Signaling Technology, 94885S,1:1000), RIPK3 (Cell Signaling Technology, 15828, 1:1000), MLKL (Abgent, AP14272B, 1:1000), GSDMD (Abcam, ab219800, 1:1000), GSDME (Abcam, ab215191, 1:1000), Tubulin (Sigma, T6199, 1:1000), GAPDH (Proteintech, 10494-1-AP, 1:5000). Original western blots are presented in Supplementary file.

The RAW264.7 cells were seeded on a 6-well plate at 1 × 10⁶ cells/well, washed three times with 1×PBS, and then lysed in protein extraction buffer (KeyGEN BioTECH, Nanjing, China) for 20 min to extract total protein. The protein concentration was determined by BCA Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, the equal 30 μg proteins from each sample were probed to SDS-PAGE and transferred to PVDF membrane (wet transfer method), and the percentage of the resolving or separating gel was 10%. The membranes were blocked with 5% nonfat milk at room temperature for 2 h and then incubated with the primary antibodies overnight at 4 °C. After incubation with the secondary antibody at room temperature for 2 h, the protein bands were visualized by ECL (Thermo Fisher Scientific, Waltham, MA, USA) and the results were analyzed by Image J. The GAPDH (Proteintech; 60004-1-Ig), RIPK1 (Proteintech; 17519-1-AP), RIPK3 (Cell Signaling Technology; 15828S), p-MLKL (Affinity; AF7420), MLKL (Abclonal; WH196063), p-RIPK1 (Abmart; TA2398S), p-RIPK3 (Abmart; TA3900S), the goat anti-rabbit IgG secondary antibody and the goat anti-mouse IgG secondary antibody were used for the Western blot analyses.

Cell culture materials were obtained from Invitrogen and fetal calf serum was from Gibco. 3-bromopyruvate, chloroquine diphosphate, 3-methyladenine (3-MA), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), Nec1 were purchased from Sigma-Aldrich. z-VAD-fmk was purchased from Calbiochem. JC-1 and Annexin V-FICT/PI assay were purchased from KeyGEN BioTECH (China). GFP-LC3 plasmid was purchased from GeneCopoeia. The following antibodies were used: LC3, Bclin-1 (MBL), Bax, Bak, Bcl-2, Mcl-1 (ProteinTech), Atg7 (Beyotime), RIPK1 (Santa cruz), RIPK3 (Cell Signaling).

Mouse tissues and treated cells were lysed by RIPA (Solarbio® Life Science, R0010) that containing proteases and phosphorylases inhibitor cocktail. After BCA assay (Solarbio® Life Science, PC0020) for determining protein concentrations, 30 μg samples were loaded to a 12% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gel and then underwent the electrophoresis under 80 V for 120 min. The gel was transmitted to a methanol activated polyvinylidene fluoride (PVDF) (Millipore®, Meck, 0.45 um, IPVH00010) membranesand blocking by 5% BSA at 4℃ for 2 h. The next day, the PVDF membrane were cut into different strips according to molecular weight of different protein like MLKL (Cell Signaling Technology™, CST37705), RIPK3 (Cell Signaling Technology™, CST95702), p-MLKL(Cell Signaling Technology™, CST37333), p-RIPK3(Cell Signaling Technology™, CST93654), LC3b II (Abcam, ab192890), p62 (Abcam, ab109012), cleaved Casp3 (Cell Signaling Technology™, 9664), GAPDH (Servicebio®, GB15002), Actin (Servicebio®, GB111364), citrullinated Histone H3 (H3cit) (Abcam, ab219407), and so on at 4℃ overnight. The membranes were washed with TBST for three times and incubated with GAR/GAM-HRP for 2 h at room temperature. At last, the membranes were washed and stained by ECL chemiluminescence.

After treatment and fixation, cardiac fibroblasts were incubated with blocking solution at room temperature. Then, diluted primary anti-α-SMA (1:100), collagen I (1:200), and collagen III (1:200) (Boster Biological Technology, Dublin, CA, USA); ki67 (1:100), PCNA (1:200) (ABclonal Technology, Wuhan, China); and the DRP1, OPA1, or RIPK3 (1:50, Cell Signaling Technology, Danvers, MA, USA) antibody were added and incubated overnight at 4 °C. After washing, the cells were incubated by IgG conjugated with Alexa Fluor 488 or Cy3 (1:500, Beyotime, Shanghai, China) without light at room temperature for 2 h followed by DAPI staining for 15 s. The cells were observed and photographed with a laser confocal microscope. The protein expression, which is considered as the fluorescence intensity, was quantified using ImageJ software.