Western Blot Analysis of TRPM1, Myc1, and ACTB

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).