Sulpho nhs lc biotin

Cell surface TGF-β receptors were visualized by cell surface protein biotinylation as described [49 (link)]. Briefly, NMuMG cells were grown to confluence, serum starved for 4–6 h, treated with 2 ng/ml TGF-β for 30 min to 1 h, and labeled with Sulpho-NHS-LC Biotin (Thermo Scientific) at 4°C for 30 min. Cells were washed with 100 mM glycine and lysed in lysis buffer. Biotinylated cell surface proteins were adsorbed to neutravidin agarose (Thermo Scientific) and analyzed by immunoblotting with antibodies to TβRI or TβRII.

After drug application, neurons were placed on ice and rinsed in cold HBSS. Neurons were then incubated in HBSS containing 1.5 mg/mL sulpho-NHS-LC-biotin (Thermo Fisher, Aalst, Belgium, cat#21335) for 20 min at 4 °C. Neurons were rinsed twice for 5 min in HBSS + Lysine 200 mM (Alfa Aesar by Thermo Fisher Scientific, Aalst, Belgium, cat#A16249) and then lysed in 300 mL RIPA buffer (Millipore, Sigma Aldrich/Merck KGaA, Darmstadt, Germany, cat#20-188) with a complete protease inhibitor cocktail (Roche, Basel, Switzerland, cat#11836170001) + phosphatase inhibitor cocktail I (Abcam, Cambridge, UK, cat#ab201112). To determine the total protein concentration by immunoblotting, 10% of the cell lysate was removed (Input). To isolate biotinylated proteins, 80% of the cell lysate was incubated with NeutrAvidin agarose (50 mL; Thermo Fisher Scientific, Aalst, Belgium, cat#29201) overnight at 4 °C on the rotating wheel. Western blots were carried out and data were quantified by comparing the ratio of biotinylated to total protein for a given culture and normalising to control untreated cultures unless stated otherwise.

This was performed as described previously13 (link). Cells were washed with ice cold PBS containing 1 mM calcium chloride (CaCl2) and 1 mM magnesium chloride (MgCl2) and incubated at 4°C for 1 h with 0.5 mg/ml Sulpho-NHS-LC-Biotin (Thermo Scientific). Cells were then incubated for 10 min at 4°C with 100 mM glycine in TBS to quench any remaining reactive biotin cross linker and lysed in ice cold modified RIPA lysis buffer with 1% mammalian protease inhibitors. Cell lysates were incubated with Streptavidin Magnetic Beads (Invitrogen) at 4°C for 2 h. Beads were washed 3 times with lysis buffer and the bound protein eluted in 1× SDS-PAGE sample loading buffer (25 mM Tris HCl, pH 6.8, containing 1% SDS, 10% glycerol, 0.001% bromophenol blue and 0.1 M dithiothreitol [DTT]). The lysate not incubated with beads was mixed with ½ volume of 3× SDS PAGE sample loading buffer and used to assess total hGLP-1R. Total and biotinylated cell surface receptors were detected by immunoblotting.