Anti cd81 sc 166029

Cells were lysed in RIPA buffer containing 5
μg.ml⁻¹ leupeptin, 1
μg.ml⁻¹ pepstatin and 1mM phenylmethylsulphonyl
fluoride. Exosomes were lysed in 8M Urea/2.5% SDS containing 5
μg.ml⁻¹ leupeptin, 1
μg.ml⁻¹ pepstatin and 1mM phenylmethylsulphonyl
fluoride. Sample loading was normalized according to Bradford relative protein
quantification and proteins separated following an electrophoretic gradient
across polyacrylamide gels. Wet electrophoretic transfer was used to transfer
the proteins in the gel onto PVDF membranes (ImmobilonP). The protein blot was
blocked for 1hr at room temperature with 5% non-fat dry milk in
PBS/0,05% Tween and incubated overnight at 4°C with the
following primary antibodies: 1:300 anti-GPC1, PIPA528055 (Thermo-Scientific);
1:300 anti-β-Actin A3854 (Sigma-Aldrich); 1:300 anti-CD81 sc-166029
(Santa-Cruz); 1:300 anti-Flottilin1 sc-25506 (Santa-Cruz). Afterwards, HRP
conjugated secondary antibodies were incubated for 1 hr at room temperature.
Washes after antibody incubations were done on an orbital shaker, four times at
10 min intervals, with 1X PBS 0.05% Tween20. Blots were developed with
chemiluminescent reagents from Pierce.