2.7. Cell Surface Biotinylation and Streptavidin Pull-Down Experiments

Cell surface biotinylation was performed according to a modified protocol described elsewhere [28]. In brief, KTC-1, KTC-Z, Nthy-ori 3-1 and Nthy-Z cells were cultured in biotin-free medium (DMEM supplemented with 10% FBS and 1 µg/mL puromycin for transduced “Z” cells) continuously for 14 days prior to commencing the experiments. Cells were grown in 10 cm Petri dishes until ~70%–90% confluent. The cells were then washed in cold PBS 2 × 30 min and incubated with 200 µg/mL biotinamidohexanoic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (B1022, Sigma-Aldrich, Steinheim, Germany) in PBS for 1 h at 4 °C with gentle shaking. Non-biotinylated controls were incubated in parallel in PBS only. Then, cells were briefly rinsed in PBS, and washed with 10 mM L-lysine (L5501, Sigma-Aldrich, Steinheim, Germany) in PBS solution 4 × 10 min to quench unbound biotin. Finally, the cells were incubated in lysis buffer (50 mM Tris, pH 6.8, with 0.2% TX-100, containing protease inhibitors as specified above), and collected in 2 mL microcentrifuge tubes to complete cell lysis and protein extraction, as described above. Cell lysates were subsequently used for SDS-PAGE and immunoblotting (see above).

For streptavidin pull-down, cells were homogenized in cold homogenization buffer (250 mM sucrose, 20 mM HEPES, 1 mM EDTA, pH 7.4), supplemented with protease inhibitors as specified above, using a hand-held homogenizer at 500 rpm for 2 × 30 s, on ice. Homogenates were cleared by centrifugation for 15 min at 10,000× g at 4 °C. The supernatant was collected and protein content was determined according to the Neuhoff assay [25].

Streptavidin-precipitation was carried out using the µMACS streptavidin kit (130-074-101; Milteny Biotech, Bergisch-Gladbach, Germany) according to the manufacturer’s protocol. Cold µMACS streptavidin MicroBeads solution was added to the cell homogenates in a ratio of 1:3 on ice and mixed by slowly pipetting up and down. The µ-column was placed in the magnetic field of the μMACS separator and prepared by rinsing it with 100 µL equilibration buffer prior to protein application, followed by two rinsing steps with 100 µL homogenization buffer (without proteinase inhibitors). The magnetically labeled complexes, i.e., streptavidin MicroBeads precipitates out of whole cell homogenates, were applied onto the top of the column matrix and washed 4 times with 100 µL washing buffer to remove non-specifically bound molecules. Elution of target molecules bound to the biotinylated probe was performed by adding 150 µL sample buffer without DTT (non-reducing) directly onto the top of the column matrix. The eluted proteins were heated for 5 min at 95 °C and stored at −20 °C until separated by SDS-PAGE.