Rabbit serum anti cd81

Conditioned media or membrane and sucrose gradient fractions were analyzed by immunoblot under denaturing and reducing conditions (0.5% SDS, 25 mM DTT). Proteins were transferred to PVDF membranes (Immobilon-P, Merck-Millipore, Darmstadt, Germany) using standard procedures and exposed to rabbit serum anti-IDE (1:40,000; #AB9210, Merck-Millipore, Darmstadt, Germany), mouse anti-PMCA (1:1000 (0.2 μg/mL); Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-flotillin 1 (1:1000 (0.45 μg/mL); Becton Dickinson, Madrid, Spain), rabbit serum anti-CD81 (1:1000 (1 μg/mL); GeneTex, Alton Pkwy Irvine, CA, USA), anti-β-actin-HRP (1:200,000 (0.015 μg/mL); Sigma-Aldrich, St. Louis, MO, USA) and followed by HRP-conjugated secondary antibodies (1:10,000; Jackson ImmunoResearch, West Grove, PA, USA). Membranes were developed with enhanced chemiluminescence reagents (ECL; Merck-Millipore, Darmstadt, Germany), and the signal was visualized with a digital camera (VersaDoc; BioRad, Madrid, Spain). The integrated optical density of the immunoreactive protein bands was measured in images taken within the linear range of the camera, avoiding signal saturation and using the Quantity One 1-D Analysis Software (BioRad, Madrid, Spain).

Cell lysates, cultured media (either directly or concentrated 20× by filter centrifugation with 10 KDa cut-off Centricon YM-10; Millipore), isolated EVs, or EV fractions were analyzed by immunoblot. Denaturing and reducing conditions (0.5% SDS, 25 mM DTT) were used to solubilize proteins prior to electrophoresis in order to detect ApoD. Proteins were transferred to PVDF membranes using standard procedures, and exposed to rabbit serum anti-human ApoD (custom made by Abyntek Biopharma against purified ApoD; Ruiz et al., 2013 (link)), goat serum anti-mouse ApoD (Santa Cruz Biotechnology), rabbit serum anti-CD81 (GeneTex), mouse anti-flotillin 1 (Becton Dickinson), or rabbit serum anti-BiP (Sigma), and followed by HRP-conjugated secondary antibodies (Santa Cruz Biotechnology). Membranes were developed with ECL reagents (Millipore) and the signal visualized with a digital camera (VersaDoc; BioRad). The integrated optical density of the immunoreactive protein bands was measured in images taken within the linear range of the camera, avoiding signal saturation.