Anti pan cadherin

Total proteins from CPC-derived cardiomyocytes were extracted by RIPA lysis buffer (Millipore, Bilerica, MA), as per manufacturer’s instructions. Proteins (30 ug) were run on a 10% SDS-PAGE gel, transferred onto PVDF membranes, and blocked. Membranes were incubated with primary antibodies: anti-NCX (1∶50000.), anti-GRP94 (1∶5000, GeneTex, San Antonio, TX), anti-GRP78 (1∶2000, Abcam, Cambridge, MA), anti-caspase-12 (1∶2000, Abcam), anti-GAPDH (1∶10000, GeneTex), anti-pan-cadherin (1∶10000, Santa Cruz Biotechnology, Santa Cruz, CA) and anti-β-actin (1∶30000, Sigma, St. Louis, MO) overnight at 4°C. Membranes were extensively washed and incubated with horseradish peroxidase-labeled immunoglobulin G (Jackson ImmunoResearch, West Grove, PA), immunoreactive bands detected by chemiluminescence methods (Millipore) and visualized on X-ray films (Kodak, Rochester, NY). Densitometric analysis was performed by Image J software (NIH, Bethesda, MD) [19] (link).

The protein profiles of M, MP, and MP@H-MnO₂-Dox-Col NP were evaluated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and coomassie brilliant blue staining (Beyotime, Shanghai, China). Western blotting was conducted to identify specific protein markers in RAW264.7 cells. After transferring the proteins onto polyvinylidene difluoride membranes, these were incubated at 4℃ overnight with anti-histone H3 (BioLegend, San Diego, CA, USA), anti-pan-cadherin (Santa Cruz Biotechnology, Dallas, TX, USA), and integrin α4 antibodies (Proteintech, Chicago, USA) and Na⁺-K⁺-ATPase (Abcam, Cambridge, UK), which was used as a reference protein.

Cells were suspended in lysis buffer (50 mM Tris-Cl, pH8.0, 150 mM NaCl, 0.5 mM MgCl₂, 10% glycerol, 1% Triton X-100, 0.1% SDS) with protease inhibitor cocktail (Roche Diagnostics) on ice for 30 min. Samples were centrifuged at 4 °C for 10 min at 12,000g. The supernatants were collected, and protein concentrations were measured using the BCA Protein Assay Kit (Beyotime) according to the manufacturer’s instructions. Protein extracts were run on a 10 or 12% SDS polyacrylamide gel before they were transferred to a polyvinylidene difluoride membrane. Membranes were blocked with 5% milk for 1 h and incubated with primary antibodies overnight, followed by incubation with the secondary antibodies for 1 h at room temperature. Antibodies are used as following: anti-ORP4L (Sigma-Aldrich, 1:1000), anti-OCRL (Santa Cruz, 1:200), anti-pan-cadherin (Santa Cruz, 1:200), anti-GM130 (Thermo Fisher Scientific, 1:3000), anti-Canexin (Cell signaling, 1:1000), anti-Cytochrome c (Santa Cruz, 1:200), anti-p-PDH (Novus Biologicals, 1:100), anti-PDH (Cell signaling, 1:1000), anti-actin (Proteintech Group, 1:3000), Goat anti-rabbit IgG (H + L), HRP conjugate antibody (Proteintech Group, 1:3000), Goat anti-mouse IgG (H + L), and HRP conjugate antibody (Proteintech Group, 1:3000).