Corresponding secondary antibody conjugated to hrp

Brain homogenates were spun at 1000×g for 10 min at 4 °C [20 (link)]. The supernatant (cortical fraction) was spun at 14,000×g for 10 min. The resulting cell pellet was resuspended and digested in RIPA buffer (Sigma) containing protease inhibitor cocktail (PIC, Sigma) for 1 h on ice. The MV pellet was resuspended and digested in a mixture of RIPA buffer (Sigma) and protease inhibitor cocktail (PIC, Sigma) for 1 h on ice. All homogenates were sonicated on ice for 90 s and spun at 14,000×g for 10 min at 4 °C for final collect of the protein lysate. Total protein was quantified by bicinchoninic assay (BCA, Pierce) and 25 µg was loaded onto a 4–20% Criterion TGX Precast Protein Gel (Bio-Rad). Protein was transferred onto a nitrocellulose membrane and probed for the neuronal marker TUJ1 (Abcam, 1:1000, ab18207) or the brain MV marker von Willebrand factor (Abcam, ab174290, 1:1000) and cyclophilin B as a loading control (Abcam, ab16045, 1:1000). Corresponding secondary antibody conjugated to HRP was used (1:5000, GE Amersham) and bands were visualized using ECL reagents (Perkin-Elmer) on an Amersham Imager 600 (GE Amersham).

Samples were loaded onto a 4–20% Criterion TGX Precast Protein Gel (Bio-Rad)^{7 (link)}. Protein was transferred onto a nitrocellulose membrane and probed for Fpn (Alpha Diagnostics, MTP11-S, 1:1000), DMT1 (Millipore, ABS983, 1:1000), Heph (Santa Cruz, SC-365365, 1:1000), TfR (Santa Cruz, sc-65882, 1:250), Tf (Abcam, ab82411, 1:1000), HA tag (Invitrogen, MA5-27915, 1:1000), or cyclophilin B (Abcam, ab16045, 1:1000) as a loading control. Corresponding secondary antibody conjugated to HRP was used (1:5000, GE Amersham) and bands were visualized using ECL reagents (Perkin-Elmer) on an Amersham Imager 600 (GE Amersham). Cellular lysate samples were normalized to cyclophilin B protein as a loading control, and then subsequently normalized to an untreated control sample within each experiment. Membrane protein samples were stained with Ponceau S and normalized to total protein as a loading control.