Goat anti rabbit hrp

Cells were washed three times with ice-cold PBS and gently scraped in lysis buffer: 50 mM Tris–HCl, pH 7.4, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.27 M sucrose, 1 mM DTT, and complete protease inhibitors (Roche). Cell lysates were snap-frozen until use. Samples were spun at 10,000g for 10 min at 4°C, and the supernatant containing the cell lysate was collected. Protein concentration was determined by Bradford assay using different concentrations of BSA as protein standards. SDS–PAGE and Western blot analyses were performed using standard protocols. Primary antibody incubation was always performed overnight at 4°C, whereas HRP-conjugated secondaries were incubated for 1 h at RT. Western blot images were acquired using a ChemiDoc gel imaging system (Bio-Rad). The following antibodies and dilutions were used in this study: rabbit anti-p62 (1:1,000; Abcam), mouse anti-NBR1 (1:1,000; Abnova 6B11), mouse anti-ubiquitin conjugates (1:1,000; Enzo FK2), mouse anti-LC3B (1:500; Nanotools), mouse anti-GAPDH (1:10,000; Sigma-Aldrich), mouse anti-tubulin III-beta (1:1,000; BioLegend TUJ1), mouse anti-puromycin (1:20,000; Merck 12D10), mouse anti-strep-tag (1:2,000; QIAGEN), mouse anti-RFP (1:1,000; ChromoTek), goat anti-rabbit HRP (1:10,000; Dianova), goat anti-mouse HRP (1:10,000; Dianova).

Antibodies used were against HSA (CD24): M1/69 fluorescein or phycoerythrin (BD Pharmingen, Erembodegem, Belgium) or allophycocyanin (Biolegend, San Diego, CA), CD69 (APC, Invitrogen Life Technologies, Darmstadt, Germany), HIV-1 Vpr (AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, Germantown, MD, USA); HIV-1 Vpr (1–46) antiserum from Dr Jeffrey Kopp (catalogue #3951) or mouse anti-HIV-1 Vpr serum [25 (link)] (kindly provided by Ulrich Schubert, Erlangen), HIV-1 p24 (AIDS Research and Reference Reagent Program; Monoclonal Antibody to HIV-1 p24 (AG3.0) from Dr Jonathan Allan) [60 (link)] or HIV-1 p24 clone KC57-RD1 (PE or FITC conjugated, Beckman Coulter, Krefeld, Germany), mouse anti-tubulin (Sigma-Aldrich, Munich, Germany), rabbit anti-NFAT (CellSignaling Merck-Millipore, Schwalbach, Germany). Secondary antibodies were IRdye 800CW Goat anti-Rabbit IgG and IRdye 680LT Goat anti-mouse IgG (Li-Cor, Lincoln, USA), Alexa-555 donkey anti-rabbit and Alexa-488 goat anti-mouse (Invitrogen) and goat anti-mouse as well as goat anti-rabbit HRP (Dianova, Hamburg, Germany). Insulin and SB213763 were from Sigma-Aldrich (Munich, Germany) and FK506 from Invitrogen.

EV pellets derived from 5.75 ml of conditioned media were resuspended in 1:1 diluted 4 × sample buffer (200 mM Tris-HCL, pH 6.8; 10% SDS; 0.4% bromophenol blue; 40% glycerol; 400 mM DTT; non-reducing conditions for CD9 and CD63 antibodies) and 20 µg of cell lysates were mixed with 4 × sample buffer. The samples were subjected to SDS-PAGE (10 or 12% polyacrylamide gels) and Western blotting using PVDF membranes. The membranes were blocked with 4% milk powder, 0.1% Tween in TBS and incubated with primary and HRP-coupled secondary antibodies. Subsequently, proteins were detected utilizing chemiluminescence (SuperSignal™ West Pico PLUS Chemiluminescent Substrate, thermo scientific, Rockford, US) and X-ray films.
The following antibodies were used: CD9 (1:2000 dilution, clone #MM2/57, Merck Millipore, Darmstadt, Germany), CD63 (1:500 dilution, #CBL553, Merck Millipore, Darmstadt, Germany), CD81 (1:1000 dilution, #B-11, Santa Cruz, Heidelberg, Germany), Syntenin (1:3000 dilution, polyclonal, ab19903, Abcam, Cambridge, UK), Calnexin (1:4000 dilution, polyclonal, SPA-865, Stressgen, San Diego, US) and HRP-coupled secondary antibodies (Goat-anti-Mouse-HRP, 115-035-003, 1:10,000; Goat-anti-Rabbit-HRP, 111-035-003, 1:10,000; Dianova, Hamburg, Germany).