Western Blot Analysis of IRS-1 and Lunapark

3T3-L1 pre-adipocytes and eWAT samples were homogenised in commercial RIPA lysis and extraction buffer (cat. no. 89900, Thermo Scientific) and protein extraction and western blotting performed as described previously [15 (link)]. Each membrane was blocked in 5% non-fat milk powder added to 0.05% TBS/T (1 × TBS, 0.05% Tween 20) and further incubated overnight with the respective primary antibody (anti-IRS-1, 1:1000 dilution [cat no. 06-248 Upstate Biotechnology, Millipore, USA] and anti-Lunapark, 1:200 dilution [cat no. NBP1–80637, Novus Biologicals, USA]) solution. Following washing in TBS/T solution, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit (1:10000) or anti-mouse (1:10000) antibody (Jackson Immuno Research, Stratech, UK). Antibody binding was detected using Super Signal West Pico Chemiluminescent substrate (Thermo Scientific) and an ImageQuant LAS 4000 machine and quantified using ImageQuant LAS 4000 software (GE Healthcare, UK). Coomassie Blue staining was used in all gels and anti-alpha-tubulin (cat no. 4074, Abcam, UK) antibody blotted on each membrane to confirm equal loading of proteins and equal transfer efficiency of samples.