Rabbit anti pan cadherin

Whole cell lysates and cell surface cell lysates from bEnd.3 and mouse primary neurons were loaded into 4%–12% Tris-glycine SDS-page gels (Invitrogen) and allowed to migrate for 1 h at 180 V. Samples were then transferred onto polyvinylidene fluoride (PVDF) membranes using an iBlot 2 Dry Blotting System (Invitrogen) on the P0 program (20 V for 1 min, 23 V for 4 min, 25 V for 2 min). PVDF membranes were then rinsed with Tris-buffered saline with 0.1% Tween 20 (TBST) and blocked for 1 h in 5% non-fat dry milk in TBST (blocking buffer). Membranes were subsequently probed overnight at 4°C with primary antibodies mouse anti-TfR H68.4 (1:500, Thermo Fisher), rabbit anti-Pan Cadherin (1:1,000, Cell Signaling Technologies), or mouse anti-tubulin (1:1,000, Cell Signaling Technologies) diluted in blocking buffer. Membranes were then rinsed multiple times with TBST before being subjected to probing with secondary antibodies diluted in TBST for 1 h at room temperature (1:10,000 diluted HRP-coupled goat anti-mouse IgG or goat anti-rabbit IgG, GE Healthcare). Following secondary antibody incubation, membranes were rinsed thoroughly with TBST and were subsequently imaged using a LICOR Odyssey Imager and quantified using Multi-Gauge v3.0.