Sepharose coupled protein a g beads

3T3-L1 adipocytes were starved in serum-free DMEM (Dulbecco's modified Eagle's medium) with 20 mM of sodium bicarbonate, 20 mM HEPES (pH 7.2) at 37°C in 5% CO₂/air for 3–8 h prior to all experiments. After insulin treatments, 3T3-L1 adipocytes were washed with 150 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 5 mM KCl, 1mM MgCl₂ (pH 7.2) and lysed in lysis buffer (Cell Signaling Technology). Western blot analyses were performed using standard protocols and densitometric analyses of immunoblots were performed using MetaMorph software. For immunoprecipitation studies protein lysates were incubated with specific antibodies for 1 h followed by incubation with Sepharose-coupled protein A/G beads (Santa Cruz Biotechnology) overnight. Beads were washed three times with lysis buffer and immunoprecipitates were resolved by SDS/PAGE and blotted as described above.

Cell lysates (500 µg) were pre-cleaned by incubation with 2 µg of rabbit immunoglobulin G (IgG) overnight under agitation and 50 µl of protein A/G-coupled Sepharose beads (Santa Cruz Biotechnology) was added to the tube and incubated for 2 h at 4 °C. After incubation, the supernatant was collected and boiled at 95~100 °C for 5 min for sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to polyvinylidene difluoride membranes, and incubated with the Gal-1 recombinant protein (500 nM) for 16 h. Membranes were probed with an anti-Gal-1 antibody followed by anti-rabbit IgG conjugated with horseradish peroxidase (HRP). The NRP1/Gal-1 complex was visualized by adding enhanced chemiluminescence (ECL) substrate to the membrane.