Odyssey fc dual mode imaging system

Protein extracts (15 µg) were subjected to 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), electrophoretically transferred to nitrocellulose membranes (Amersham Hybond ECL, GE Healthcare), and blocked in 3% BSA/PBS at RT for 1 hour. Primary antibodies were incubated overnight at manufacturer recommended concentrations. Immunoblots were detected with the ECL Plus Western Blotting Detection System (Amersham Biosciences, Piscataway, NJ) or using near-infrared fluorescence with the ODYSSEY Fc, Dual-Mode Imaging system (Li-COR). Additional exposure images are provided in Supplementary data. Expression levels were evaluated by quantification of relative density of each band normalized to that of the corresponding β-actin or GAPDH band density.

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)}.

Western blots were carried out as previously described^{60 (link)}. Details of the primary antibodies (anti-TRPM1, -Myc1, and -ACTB antibodies) are shown in Supplementary Table 2. Despite multiple attempts with varying conditions, Western blot using the anti-LRIT3 antibody was unsuccessful resulting in non-specific reactions with the canine samples. We therefore chose Myc-tags for the identification of LRIT3 products in vitro. Briefly, retinal or cell lysates were quantitated using the BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). Experiments were done in triplicate with 30 μg of protein loaded per lane. The immunoblot was scanned on the Odyssey Fc Dual-Mode Imaging System (LI-COR, Lincoln, NE) and normalized against β-actin using the LI-COR Image Studio Software (LI-COR). For each sample, the average of the three β-actin-normalized values per each antibody was compared with that of the corresponding normalized and averaged value of the WT sample and expressed as fold-changes. Statistical significance was calculated by an unpaired t-test using GraphPad (GraphPad Software, San Diego, CA).

Whole-cell extracts were prepared by scraping cells directly into 2× SDS Sample Buffer with 10 mM DTT. Samples were syringed to shear DNA or sonicated and boiled at 95°C for 5 min prior to SDS-PAGE on 4–12% precast gradient gels (Invitrogen). Transfer onto Immobilon-P or Immobilon-FL membranes was carried out using the XCell IITM Blot Module according to the manufacturer's instructions. Membranes were blocked in PBS, 0.1% Tween-20, 5% dried milk, and processed for immunoblotting with the primary antibodies indicated in the figures. Secondary antibodies used were HRP-conjugated, or IRDye^® 680RD- or 800CW-conjugated for quantitative fluorescence measurements on an Odyssey^® Fc Dual-Mode Imaging System (LI-COR Biosciences). For cell fractionation experiments, cells were harvested and treated at 4°C with 40 µg ml⁻¹ digitonin in 10 mM Tris–HCl (pH 7.4), 100 mM NaCl, 25 mM MgCl₂, 1 mM Na₃VO₄, 1 mM NaF and EDTA-free protease inhibitor cocktail (Roche). Cells were disrupted by 10 passages through a 25-gauge needle (whole-cell extract). Cell nuclei were pelleted by centrifugation at 3000 r.p.m. for 10 min in a microfuge at 4°C. The resulting supernatant was re-centrifuged at 13 000 r.p.m. for 15 min to yield mitochondrial (pellet) and cytoplasmic (supernatant) fractions.