0.45 m nitrocellulose membrane

Twenty micrograms of ECV membrane proteins were separated on polyacrylamide gels as described above. For the detection of E. chaffeensis Dnak and p28‐Omp 19 proteins, the above-described electrophoresed proteins were transferred onto a 0.45 µm nitrocellulose membrane (Thermo Fisher Scientific, Rockford, IL) and subjected to blotting using p28 monoclonal antibodies and polyclonal rabbit antisera raised against recombinant E. chaffeensis protein DnaK, respectively (Zhang et al., 2013 (link)). For the detection of host membrane proteins Rab5 and Lamin B, anti-rabbit Rab5 and anti-rabbit Lamin B antibodies (1:1000) (Cell Signaling Technology, 110 Danvers, MA) were used, respectively. Secondary antibodies conjugated with horseradish peroxidase (Sigma-Aldrich, St. Louis, MO, USA) were used. ECL Western blotting detection reagents (Amersham, Buckinghamshire, UK) were used for the signal detection.

Samples were run on Bolt 4–12% Bis-Tris Plus gels (Thermo Fisher Scientific, Waltham, MA, USA) and transferred to 0.45 µm nitrocellulose membrane (Thermo Fisher Scientific, Waltham, MA, USA) using a Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad, Hercules, CA, SA). Five percent (w/v) non-fat dry milk in Tween-PBS (PBS with 0.1% Tween 20) was used to block membranes for at least 1 h. Membranes were probed with mouse anti-myc tag antibody (2276; Cell Signaling Technology, Danvers, MA, USA) or mouse anti-V5 antibody (AB27671; Abcam, Cambridge, UK) in 2% (w/v) non-fat dry milk in Tween-PBS over-night at 4 °C. Following three Tween-PBS washes, the membranes were incubated with anti-mouse secondary antibody conjugated with horseradish peroxidase (A16072; Life Technologies, Inc.) or goat anti-mouse DyLight 800 (SA5-35521, Thermo Fisher Scientific, Waltham, MA, USA) in 2% (w/v) non-fat dry milk in Tween-PBS for 1 h. Membranes were again washed three times with Tween-PBS; horseradish peroxidase signal detected on Bio-Rad Chemidoc system (Bio-Rad, Hercules, CA, USA) using chemiluminescence detection reagents (Thermo Fisher Scientific, Waltham, MA, USA) or alternatively on Odyssey CLx (LI-COR Biosciences, Lincoln, NE, USA) to detect fluorescence.

To test ALOX15 expression aliquots (2–20 µL) of the lysis supernatants were analyzed by SDS-PAGE on a 7.5% polyacrylamide gel. Separated proteins were transferred to a 0.45 µm nitrocellulose membrane (Thermo Scientific GmbH, Schwerte, Germany) by a wet blotting method (ProSieve Ex Western Blot Transfer Buffer 10x, Biozym Scientific GmbH, Hessisch-Oldendorf, Germany). The membranes were blocked with blocking solution (10x BlueBlock PF for Blotting, SERVA Electrophoresis GmbH, Heidelberg, Germany), washed three times with PBS/0.3% TWEEN 20 and were finally incubated with an anti-His-HRP antibody (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) for 1–2 h at room temperature. After several steps of washing, the membrane was stained using the SERVALight Polaris CL HRP WB Substrate Kit (SERVA Electrophoresis GmbH, Heidelberg, Germany) for 5 min at room temperature. Chemiluminescence was quantified using the FUJIFILM Luminescent Image Analyzer LAS-1000plus (Fujifilm Europe GmbH, Düsseldorf, Germany).

Samples were incubated with SDS loading buffer at 95 °C for 10 min followed by centrifugation for 1 min at 10,000× g. Then 20 µL per lane were loaded on a 10% SDS-polyacrylamide gel (Hoefer SE260 system, Serva, Heidelberg, Germany). Blotting was carried out onto a 0.45 µm nitrocellulose membrane (Thermo Fisher Scientific, Schwerte, Germany) using a semi-dry blotting system (V20-SDB, Scie-Plas, Cambridge, UK) following standard protocols [69 ]. The membrane was blocked with 10% (w/v) non-fat milk in TBS before incubation with the VP-specific primary antibody B1 (1:100, anti-AAV VP1/VP2/VP3 mouse monoclonal supernatant (Progen Biotechnik, Heidelberg, Germany)) in blocking buffer for 1.5 h. Detection was performed after incubation with a secondary anti-mouse IgG linked to a horseradish peroxidase (1:5000, anti-mouse IgG, HRP-linked antibody, Cell Signaling Technology, Leiden, The Netherlands) by luminescence detection (Pierce ECL western blot substrate, Thermo Fisher Scientific).

Cell pellets from rAAV production (1× 100 mm dish) were resuspended in 100 µL PBS and 5× SDS loading buffer. Samples were incubated at 95 °C for 10 min, centrifuged and 20 µL per lane were loaded on a 10% SDS-polyacrylamide gel (Hoefer SE260, Kleinbittersdorf, Germany). Samples were blotted onto a 0.45 µm nitrocellulose membrane (Thermo Fisher Scientific) using semi-dry electrophoretic transfer (V20-SDB, Scie-Plas, Cambridge, UK). After blocking the membrane with 10% (w/v) non-fat milk in TBS, the membrane was incubated simultaneously for 1.5 h with the B1 antibody (mouse monoclonal, supernatant, 1:100, Progen, Heidelberg, Germany) and an anti β-Actin antibody (8H10D10, mouse monoclonal, 1:1000, Cell Signaling Technology, Frankfurt am Main, Germany). After incubation with an anti-mouse IgG, HRP-linked antibody (1:5000, Cell Signaling Technology), blots were imaged by luminescence detection (Pierce ECL Western Blot Substrate, Thermo Fisher Scientific).