Ripa cell lysis buffer system

BMMSCs were cultured and treated as described previously, and proteins were harvested with RIPA cell lysis buffer system (Cell Signaling Technology, USA) supplied with phosphatase inhibitors. Total protein concentrations were quantified by BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Equal amounts of proteins (20 μg) were separated on 10% SDS-PAGE for protein electrophoresis and transferred to 0.2 μm polyvinylidene fluoride (PVDF) membranes, which were blocked with 5% nonfat dry milk for 1 h at room temperature and incubated at 4°C overnight with primary antibodies to mouse Runx2, RIPK1, RIPK3, MLKL, phosphor-RIPK3 (p-RIPK3), phosphor-MLKL (p-MLKL), cleaved caspase-3, and β-actin (1 : 1000; Abcam, Cambridge, UK) and phosphor-RIPK1 (p-RIPK1; 1 : 1000; Cell Signaling Technology, USA). After washing three times in PBST and incubated for 1 hour with HRP-conjugated second antibody at room temperature (1 : 2000, Abcam, Cambridge, UK), immunoreactive protein was detected using enhanced chemiluminescence reagents (Millipore, USA) and immunoblots were quantified with ImageJ software. MC3T3-E1 were cultured with osteogenic differentiation induction medium and treated as described previously to verify EtOH-induced osteoblast necroptosis.

Total protein was extracted from kidney and mouse NRK-52E cells using the RIPA cell lysis buffer system (Cell Signaling Technology, USA), supplemented with phosphatase inhibitors, and was quantified by BCA-protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). An aliquot (20 μg) of the proteins was separated by 10% SDS-PAGE for protein electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore). Following blocking in 5% nonfat dry milk for 1 h at room temperature, the membrane was incubated at 4°C overnight with primary antibodies. The following antibodies were used: mouse RIPK1, p-RIPK3/RIPK3, p-MLKL/MLKL, IL-1β, anti-active caspase-3, phosphor-inhibitor κB α (p-IκBα), phosphor-inhibitor of kappa B kinase α/β (p-IKKα/β), GAPDH (all antibodies dilution ratios are 1 : 1000; Abcam, Cambridge, UK), and rabbit p-RIPK1. After washing three times with phosphate-buffered saline with Tween-20 (PBST), the membrane was incubated with HRP-conjugated secondary antibody (1 : 2000, Abcam, Cambridge, UK) at room temperature for 1 h. Bands were quantified using ImageJ software and normalized to GAPDH.