Anti abcg2 bxp21

In order to confirm the presence of EVs, the EV surface markers CD63 [70 (link)] and CD81 [71 (link)] were analyzed by Western blot. For this purpose, the EV-containing elution fraction (see Section 4.5) was concentrated using Amicon^® ultra filters 2 mL 3 kDa (Merck, Darmstadt, Germany). Concentrates were added with RIPA lysis buffer supplemented with pefabloc (1 mg/mL), aprotinin (1 µg/mL), pepstatin A (1 µg/mL), and leupeptin (5 µg/mL) as protease inhibitors. To compare the expression of ABCC2 and ABCG2 in different cell types, cellular proteins were purified as described in 4.4. Proteins were separated by SDS-PAGE and blotted onto a PVDF membrane. Subsequently, membranes were blocked with 5% bovine serum albumin for at least 1 h and incubated overnight with the primary antibodies anti-CD63 (sc-5275, 1:200 in blocking buffer), anti-ABCG2 (BXP21, 1:100 in blocking buffer) from Santa Cruz Biotechnology (Heidelberg, Germany), anti-CD81 (555675, 1:200 in blocking buffer) from BD Biosciences (Heidelberg, Germany), or anti-ABCC2 (M2-III-6, 1:100 in blocking buffer, Enzo Life Sciences, Farmingdale, USA). Mouse IgG HRP-Linked Whole Antibody (1:2000, Cytiva, Marlborough, MA, USA) was used as the secondary antibody. Immunoreactive bands were detected by chemiluminescence using the ChemoStar touch device (Intas Science Imaging Instruments GmbH, Göttingen, Germany).