Hrp conjugated second antibody

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 OS cells using RIPA buffer (Beyotime, Shanghai, China) and quantified using BCA assay kit (Bio-rad, China) as per the manufacture's instruction. Then total protein (40 μg) from each sample was separated using SDS-PAGE and transferred to a PVDF membrane. The membranes were probed with anti-E-cadherin antibody (Abcam, ab76055), anti-Vimentin antibody (Abcam, ab8978), anti-N-Cadherin antibody (Abcam, ab98952), anti-Snail antibody (Abcam, ab117866), anti-ZEB1 antibody (Abcam, ab181451) and anti-β-actin antibody (Abcam, ab8224) after blocked with 5 % non-fat milk. Then the membranes were further probed with HRP-conjugated second antibody (Abcam, ab6728). β-actin was used as an internal control and protein bland was detected with enhanced chemiluminescence (Amersham Pharmacia). Densitometry of Western blots was quantified using NIH ImageJ software (Bethesda, USA). Blotting images and densitometry results were representative from 3 repeats.