β pix

For co-immunoprecipitation (co-IP) done with HEK293T cells, the cells were plated to reach 60–70% confluency for the day of transfection and after 24 hours of transfection, the cells were collected. For co-IP done with neurons, DIV8–9 cultures were used. For both cell lines and primary neurons, the samples were treated either with 50nM leptin or vehicle for indicated durations. The samples were lysed with cold TNE buffer (1% Nonidet P-40, 140 mM NaCl, 5 mM EDTA, and 50 mM Tris-HCl, pH 8.0) that was supplemented with cOmplete protease inhibitor cocktail and phosphatase inhibitor cocktail 2 and 3 (Sigma Aldrich) on ice for 20–30 min and the lysates were centrifuged for 10 min at 16,000g [32 (link)]. 10% of the lysate was separated as input and the rest of the lysate was incubated with c-myc (Sigma-Aldrich #4439, 1:100) and V5 (Cell Signaling #13202, 1:100) antibodies for HEK293T cells, and β-PIX (Millipore #07–1450-I, 1:100) antibody for neurons for 2 hours at 4°C, rotating. Then, they were incubated with Protein A/G magnetic beads for another 2 hours at 4°C, rotating. Supernatant was discarded and the beads were washed four times, each for 10 min, with TNE buffer. The samples were eluted with 50mM DTT (Fisher Scientific) and NuPage LDS Sample Buffer (Life Technologies), by heating at 75°C for 10 min and analyzed by western blotting.