Odyssey clx imagining system

A mixture of TSP1 and rh-CD47p, with or without the monoclonal TSP1 antibody (mAb 301221, Novus Biologicals, Littleton, CO, USA), was prepared in a cell-free setting and incubated at 37 °C for 45–60 min. Upon incubation, TSP1 and rh-CD47p mixture samples were subject to electrophoresis on gradient 4–20% Mini-PROTEAN TGX precast gels (BioRad, Hercules, CA, USA) for approximately 1 h at 200 V, followed by transfer to a nitrocellulose membrane (BioRad, Hercules, CA, USA) at 4 °C with a constant current of 150 mA for 2 h. Membranes treated with Odyssey blocking buffer (LI-COR, Lincoln, NE, USA) at room temperature for 1 h were exposed to a primary antibody against TSP1 (A6.1, Abcam, Cambridge, MA, USA, 1:500) at 4 °C overnight and subsequent incubation with a red-fluorescence-labeled secondary antibody (926-68050, LI-COR, Biosciences, Lincoln, NE, USA, 1:10,000) at room temperature for 1 h. Membranes were reprobed with a primary antibody against rh-CD47p (EPR21794, Abcam, Cambridge, MA, USA, 1:500) at 4 °C overnight and a green-fluorescence-labeled secondary antibody (926-32213, LI-COR, 1:10,000) at room temperature for 1 h. Images were captured by an Odyssey CLx imagining system (LI-COR, Biosciences, Lincoln, NE, USA).

Bacterial whole-cell and supernatant samples were prepared as before (7 (link), 8 (link)). Briefly, cultures were grown to an optical density at 600 nm (OD₆₀₀) of 0.5, and the OD 0.5 cultures were pelleted by centrifugation and resuspended in 50 μl of Laemmli buffer for the whole-cell samples. Supernatant samples were obtained by trichloroacetic acid (TCA) precipitation of cell-free supernatants (7 (link), 8 (link)). Protein samples were analyzed by SDS-PAGE, transferred to a nitrocellulose membrane, probed with polyclonal anti-CpaA (1:1,000) (8 (link)) and/or monoclonal anti-RNA polymerase (1:2,000; BioLegend). Western blots were probed with IRDye-conjugated secondary antibodies and visualized with an Odyssey CLx imagining system (Li-COR Biosciences, Lincoln, NE).