Journal: Theranostics

Article Title: GRP78-targeted ferritin nanocaged ultra-high dose of doxorubicin for hepatocellular carcinoma therapy

doi: 10.7150/thno.30867

Figure Lengend Snippet: SP94 peptide specifically binds to GRP78 in HepG2 cells. (A) The experimental procedure of identifying the membrane receptor of SP94 peptide. (B) SDS-PAGE analysis of the membrane proteins on HepG2 cells bound to biotin-SP94. The biotinylated FPWFPLPSPYGN peptide, which is not able to bind to HepG2 cells, was used as a negative control. Bands were identified by Coomassie Blue staining. (C) The peptide sequence of the target protein was deduced by mass spectrum analysis. (D) Protein immunoprecipitated from HepG2 cell surface by biotin-SP94 peptide was recognized by mouse anti-GRP78 mAbs and visualized with HRP-coupled anti-mouse IgG. (E) The binding activity of biotin-SP94 was reduced after GRP78 gene knockdown in HepG2 cells. (F) The binding activity of biotin-SP94 was enhanced when GRP78 was overexpressed in mouse 3T3-L1 cells.

Article Snippet: After blocking with non-fat dry milk, the nitrocellulose membrane was incubated with a 1:1,000 dilution of mouse anti-GRP78 monoclonal antibody (mAbs, Rockland) and a 1:3,000 dilution of mouse anti-β-actin monoclonal antibody (mAbs, Sigma) overnight at 4°C, and developed with a 1:3,000 dilution of HRP-conjugated anti-mouse IgG.

Techniques: SDS Page, Negative Control, Staining, Sequencing, Immunoprecipitation, Binding Assay, Activity Assay