Enhanced ecl kit

Total proteins were extracted and separated by 10% SDS-polyacrylamide gel electrophoresis, and then transferred onto a nitrocellulose membrane (Millipore, Burlington, MA, USA). The membrane was incubated in 5% low-fat milk in Tris-buffered saline with 0.1% Tween-20. Subsequently, the membranes were incubated with the rabbit antibodies against human E-cadherin, N-cadherin, vimentin, p-AKT, AKT, p-ERK, ERK, PI3K, and GAPDH (Servicebio, Wuhan, China) at 4 °C overnight and were then incubated with secondary antibodies at 1:5000 dilution. Finally, signals were visualized by an enhanced ECL kit (Millipore, Burlington, MA, USA) (original western blot see Figures S7 and S8).

After appropriate treatment, cells were lysed for total protein extraction by RIPA Lysis Buffer (Beyotime) containing protease and phosphatase inhibitors, and the protein concentration was measured using a BCA assay kit (Beyotime, Beijing, China). Then, 20 μg of whole proteins from each group was loaded into 10% or 12% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS-PAGE) gels for separation and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with TBST supplemented with 5% nonfat dried milk and incubated with primary antibodies at 4 °C overnight. Primary antibodies against LC3B (1:1000, CST), p62 (1:2000, Abcam), Beclin-1 (1:1000, CST), BAX (1:1000, Abcam), Bcl-2 (1:2000, Abcam), caspase-3 (1:1000, CST), PI3K (1:500, CST), p-PI3K (1:500, CST), AKT (1:500, CST), p-AKT (1:500, CST), mTOR (1:500, CST), p-mTOR (1:500, CST), Collagen II (1:500, Abcam), MMP-3 (1:1000, CST) and MMP-13 (1:1000, CST) were used in this study. After washing the membranes three times, the membranes were incubated with the relevant secondary antibodies for 1 h at room temperature. The protein bands were evaluated by an enhanced ECL kit (Millipore, USA) and a Chemiluminescence Imaging System (Tanon-4800, Shanghai, China), and the data were analyzed by ImageJ software (National Institutes of Health, USA).

DiI-labeled sLip and SP-sLip (DiI 0.4 mg mL⁻¹, phospholipid 10 mg mL⁻¹) were injected via the tail vein of BALB/c mice. Whole blood was sampled with EDTA anticoagulant in Protein LoBind tubes at 1 h after administration, and plasma was separated by centrifugation at 1000 × g for 10 min. Liposomes were normalized by detecting the fluorescence intensity of DiI (Ex 555 nm, Em 575 nm). The isolated protein coronas absorbed on liposomes (procedure was the same as in vitro) were subjected to Western Blotting analysis. Briefly, after separation by 4–20% SDS-PAGE, the samples were transferred to PDVF membrane, and blocked by 5% nonfat milk in PBST (PBS containing 0.1% Tween-20) for 1 h at room temperature. Primary antibodies (1:1000 for anti-ApoJ, 1:2000 for anti-ApoE, 1:5000 for anti-ApoA1) were incubated with the membrane overnight at 4 °C. Following by three times wash with PBST, the membranes were incubated with horseradish conjugated secondary antibodies (1:2000 anti-goat IgG for ApoJ, 1:5000 anti-rabbit IgG for ApoE and ApoA1) at room temperature for 1 h. After six washes of PBST, enhanced ECL kit (Millipore) was used to react with the membrane, and the signal was imaged (ChemiScope 6000, Clinx Co. Ltd) (see Supplementary Figs. 12–14). The data were analyzed by Image J software.