Immunoprecipitation and Immunoblotting of Cellular Proteins

Cell lysate (1 mg) was incubated for 1 h with IgG and 10 μg of protein A/G beads [24 (link)]. The lysate was then incubated overnight at 4 °C with 10 μg of anti-ARG2 (CST.) and anti-ARL1 (SCB) antibodies or nonspecific IgGs. Following this, the lysate was incubated once more for 1 h at RT with 20 μg Pierce^TM protein A/G magnetic beads (Thermo Fisher Scientific Inc.). The samples containing the beads were placed in a magnetic separation rack and then the supernatants carefully removed. Pellets were washed with 1 mL of the lysate buffer (10 Mm Tris-HCl, pH7.4, 150 mM NaCl, 5 Mm EDTA, 0.1% Triton X-100). The immunoprecipitated proteins were extracted by 10 min boiling with 2x SDS-PAGE reducing sample buffer 40 μL and detected by immunoblot with anti-HIF1A or anti-Slug antibodies