Streptavidin agarose beads suspension

Cells expressing wild-type IR and IGF1R or chimeric receptors (IR/IGF1R or IGF1R/IR) were serum starved in DMEM supplemented with 0.1% BSA for 3 h, followed by the stimulation with 100 nM insulin (for IR and IR/IGF1R) or 100 nM IGF-1 (for IGF1R and IGF1R/IR) for 0, 30 and 120 min. The cells were rinsed once with ice-cold PBS, followed by 1 ml 0.3 mg ml⁻¹ sulfo-NHS-Biotin (ThermoFisher Scientific) labelling at 4 °C for 30 min, and the labelling reaction was quenched by the addition of ice-cold 100 mM glycine (pH 3) for 10 min. Cells were washed twice with ice-cold PBS, and lysed in RIPA buffer supplemented with 10 mM glycerophosphate, 10 mM NaF, 0.1 mM sodium orthovanadate and 1% protease inhibitor cocktail (Sigma). Biotinylated surface proteins were enriched by incubating 100 μg total protein lysates with 10 μl streptavidin-agarose beads suspension (50% slurry, ThermoFisher Scientific) in 800 μl total volume on a rotator at 4 °C for 1 h. Subsequently, beads were washed with RIPA lysis buffer three times and bound proteins were liberated from the beads by boiling in 1 × SDS-PAGE loading buffer. The biotinylated surface protein fractions as well as total protein lysates were resolved on SDS-PAGE gels, transferred to PDVF membrane and immunoblotted using IRβ and IGF1Rβ antibodies.

The clarified glioblastoma homogenate was used to generate two fractions, either free or enriched with the biotin-containing proteins. The level of glioblastoma homogenate proteins was set to 1 µg/ml with Dulbecco’s Phosphate Buffered Saline (DPBS; D8537, Sigma). The biotin-containing proteins were extracted from lysates by the already used method [38 (link)]. Briefly, 150 µl of diluted lysate was incubated with 500 µl of streptavidin-agarose beads suspension (Thermo-Fisher) at room temperature for one hour. Subsequently, the suspension was centrifuged at 3000×g for 5 min, and supernatant-1 was collected. The sediment was washed twice with DPBS to remove unspecifically interacting proteins; those were collected and combined with supernatant-1. The generated supernatant-1 was depleted of biotin-containing proteins. The biotin-containing proteins were eluted from a complex with streptavidin agarose beads with DPBS supplemented with biotin (100 µg/ml). During incubation at room temperature for 30 min. After centrifugation of suspension at 3000×g for 5 min, supernatant-2 was collected. The proteins in the diluted glioblastoma homogenate, supernatant-1, and supernatant-2, were precipitated with acetone and collected by centrifugation at 12,000×g for 12 min. Supernatants were discarded, and air-dried pellets were resuspended in DPBS and used for further analysis.