Hrp coupled goat anti mouse igg

NHP-derived BEC were lysed using ice-cold radio-immunoprecipitation assay (RIPA) or cell lysis buffer containing protease inhibitor cocktail (ThermoFisher) centrifuged at 15,000g for 15 min, and supernatant fractions were collected. Samples were added to SDS and loading buffer then denaturized by heat at 95 °C for 5 min. Denaturized samples were loaded into 4–12% Tris–Glycine SDS-page gels (Invitrogen), and left to migrate for 1 h at 180 V. Samples were then transferred onto polyvinylidene fluoride (PVDF) or nitrocellulose 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 first probed overnight at 4 °C with primary antibodies in blocking buffer (anti-ITM2A polyclonal AF4876 and 18306-1-AP, EmGFP A11122, anti-HA 901509, anti α-tubulin T9026) and then probed with secondary antibodies diluted in TBST for 1 h at RT (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, imaged using a LICOR Odyssey Imager and bands quantified using Multi-Gauge v3.0.