Image studio software

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).

The images were quantified using the default ‘Analyze particles’ plugin in ImageJ. Additionally, the co-localised pixels were identified, and a profile was generated using an unsupervised ImageJ plugin algorithm called ‘colocalization’, which was developed by Pierre Bourdoncle (Institut Jacques Monod, Service Imagerie, Paris; 2003–2004). The western blots were quantified using the LICOR Image Studio software (LICOR, US) and the values were processed, analysed and plotted using Excel program from the Microsoft Office suite.
The statistical significance levels for comparisons between two groups were estimated using one-tailed or two-tailed t-tests. For the experiment involving ATG7 and ATG10 KD, one-tailed t-test was used to assess the statistical significance of the data. For all the other experiments in this study, a two-tailed t-test was used to assess the statistical significance of the data. In this paper, the value of p < 0.05 was considered statistically significant and the conventions used to depict the results across this article are as follows: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 and Error bars represent standard deviations unless otherwise mentioned.

Protein expression in adipose tissue was measured by immunoblotting as we described^{69 (link),76 (link),77 (link)}. Fat tissues were homogenized in a modified radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with 1% protease inhibitor mixture and 1% phosphatase inhibitor mixture (Sigma-Aldrich, St. Louis, MO). Tissue lysates were resolved by SDS-PAGE. Proteins on the gels were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA), which were then blocked, washed, and incubated with various primary antibodies, followed by Alexa Fluor 680-conjugated secondary antibodies (Life Science Technologies). The blots were developed with a Li-COR Imager System and analyzed with Li-COR Image Studio Software (version 2.1, Li-COR Biosciences, Lincoln, NE). The following primary antibodies were used: UCP1 (1:1000, Abcam, ab23841), TH (1:1000; AB152, EMD Millipore, Temecula, CA), NT-3 (1:1000, AF-267-NA, R&D Systems), pHSL (1:1000, 4126S, Cell Signaling Technology), HSL (1:1000, 4107S, Cell Signaling Technology), and α-tubulin (1:1000, Advanced BioChemicals, ABCENT4777). The antibody information is listed in Supplementary Table 1.
Tissue and serum NT-3 content was measured by ELISA (ABCE-EL-M2438, Advanced BioChemicals, Lawrenceville, GA).

In vitro glycosylation assays were conducted as previously described [1 (link)]. Enzymes (200 nM of NleB1, NleB, or SseK1) were incubated in 50 mM Tris-HCl buffer pH 7.4, 1 mM UDP-GlcNAc, 10 mM MnCl₂, and 1 mM DTT with 1 mM GAPDH in the presence or absence of serial dilutions of avasimibe. After a 2 h incubation at room temperature, samples were blotted with anti-R-GlcNAc and anti-His tag monoclonal antibodies (Abcam, Cambridge, MA, USA). LI-COR Image Studio software (LI-COR Biosciences, Lincoln, NY, USA) was used to measure signal intensities, and inhibition was estimated by measuring the relative reduction in substrate glycosylation.

Central brains (optic lobes manually removed) were homogenized in 4 ml 1xLB (loading buffer) per brain. Lysates were loaded into 4–20% Tris-glycine gels (Lonza, Allendale, NJ, USA) and transferred to Immobilon-FL (Millipore, Billerica, MA, USA). Blots were probed with rabbit anti-Draper (1 : 1000, kind gift from Marc Freeman) and sheep anti-tubulin (Cytoskeleton, Denver, CO, USA; no. ATN02). Blots were incubated with primary antibodies overnight at 4 °C, washed 3 × with 1xPBS+0.01%Tween-20, and then incubated with secondary antibodies (713-625-147 and 711-655-152, from Jackson Immunoresearch, West Grove, PA, USA) for 2 h at room temperature. Blots were then washed 3 × with 1xPBS+0.01% Tween-20 and 1 × with 1xPBS. Blots were imaged on LI-COR Odyssey CLx Quantitative Western Blot Imaging System, and data were quantified with Li-COR Image Studio software (Li-COR, Lincoln, NE, USA).