Odyssey infrared detection system

Puromycin-selected HeLa or COS-1 cells were seeded in 6-well plates and transfected with 0.3 ug NL4-3 (HeLa) or 1 ug pSARMX proviral vector using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions. On day 2 post-transfection, cell pellets and culture supernatants were harvested. Culture supernatants were clarified by low speed centrifugation and filtered through a 0.2 µm filter, then layered onto a 20% sucrose cushion in PBS and centrifuged at 20,000 g for 2 hours at 4°C using Sorvall Discovery M-150 SE micro-ultracentrifuge (Thermo Electron Corporation, Waltham, MA, USA). Virion pellets and corresponding cell pellets were dissolved in Laemmli sample buffer (Bio-Rad, Hercules, CA, USA). Virion and cell lysates were separated on 12% polyacrylamide gels and subjected to Western blotting. HIV-1 Gag detection was performed with mouse anti-p24 monoclonal CA-183 antibody (provide by Bruce Chesebro and Kathy Wehrly through the NIH AIDS Research and Reference Reagent Program). Rabbit anti-p27 polyclonal antibody [41] (link)was employed for detection of M-PMV Gag. IRDye goat anti-mouse and IRDye goat anti-rabbit secondary antibodies used for Western blots were obtained from Li-cor Biosciences (Lincoln, NE, USA). Blots were developed and analyzed using the Li-Cor Odyssey infrared detection system.

Each strain harboring the His-tagged constructs (Table 3) was grown in static overnight biofilm cultures as described above. For plasmid maintenance, ampicillin was added. Culture supernatants were subsequently collected, filtered sterilized, and TCA precipitated. The total protein pellets were resuspended in 1 ml NP-40 with protease inhibitor cocktail (Roche Diagnostics GmbH). Samples were precleared using protein A/G plus agarose beads (Pierce), followed by immunoprecipitation with a mouse anti-His affinity resin (GenScript) or a negative-control mouse IgG antibody (Santa Cruz). Samples were incubated overnight at 4°C with rotation. On the following day, protein A/G plus agarose beads were added to the negative-control IgG samples, and the mixture was incubated for 1 h at 4°C with rotation. Afterwards, the beads or resin samples were pelleted, washed six times, and boiled in acidified Laemmli lysis buffer as previously described for adherence proteins (91 (link)). After boiling, the samples were neutralized with basic Laemmli lysis buffer. Samples were run on a 4 to 20% SDS-PAGE gel (Bio-Rad), and Western blot analysis was performed as previously described (30 (link)) using a primary anti-His antibody (Qiagen) and a secondary Alexa Fluor 700 goat anti-mouse immunoglobulin antibody. The Western blots were scanned using an Odyssey infrared detection system (Li-Cor).

Equal amounts of protein (30–80 μg/lane) were resolved by a 7.5, 10 or 12.5% SDS-PAGE. Subsequently, proteins were transferred to a nitrocellulose membrane using a Tankblot Eco-Mini system (Biometra, Göttingen, Germany) and further processed as described previously [26 (link)]. Primary antibodies for antigen detection are listed in the Additional file 1: Table S4. IRDye800CW-conjugated goat anti-rabbit secondary antibody or IRDye680RD-conjugated donkey anti-mouse secondary antibody were applied for visual detection using the Odyssey Infrared Detection system (LI-COR Biotechnology, Bad Homburg, Germany). β-ACTIN or HSP90 expression served as invariant controls.
Recruitment of iBAT was calculated as UCP1 content in total iBAT: [UCP1 expression * dilution factor for protein content * (total iBAT in mg / weighed portion of iBAT for protein isolation in mg)] (normalised values were used).
Relative UCP1 capacity index was calculated as follows: [(relative protein expression * total iBAT tissue (mg))/body mass (mg)] * 100%.