Western Blot Analysis of Tissue Proteins

Brain, eyes or retinas from ≥three animals were homogenized in cold RIPA buffer containing proteases inhibitors (Roche Complete Mini-EDTA free protease inhibitors, 100 μM leupeptin, 100 mM NaVO4, 20 mM NaF) using a BeadBug™ Microtube Homogenizer (Benchmark Scientific Model D1030E) as in^{10 (link)}. 50 μg extract samples were electrophoresed on 4–12% SDS- polyacrylamide gels and transferred to nitrocellulose membranes (Li-COR #926–31090). Membranes were incubated in Odyssey® Blocking Buffer (Li-COR, #P/N 927–50000) for 1 h at room temperature, treated with primary antibody (Supp. Table S2) in Blocking Buffer +0.1% Tween 20 for 12–6 h at 4 °C and incubated for 1 h at room temperature in secondary antibody (goat anti-rabbit IRDye 800CW or goat anti-mouse IRDye680RD; LiCOR). Membranes were scanned on Odyssey Fc Dual-Mode Imaging System (Li-COR) and data quantified using Image Studio Software (Li-COR), according to manufacturer’s protocols. Actin was used as a reference protein for quantitative immunoblots. All commercially available antibodies used for immunoblots and immunofluorescence experiments are listed in Supp. Table S2. Affinity purified rabbit polyclonal anti-Ndr2 antibody was generated using an Ndr2-specific peptide antigen (QPVPNTTEPDYKSK, corresponding to amino acids 421–434) (YenZym Antibodies LLC, San Francisco, CA), as previously described^{29 (link)}.