Chemidoc xrs station

Conditioned supernatants of multiple myeloma NCI-H929 cells were obtained by washing the cells in PBS and culturing them in Protein-Free Hybridoma medium for 72 h. Supernatants were collected and centrifuged at 12,000 × g for 30 min to remove cell debris. They were further concentrated using Amicon Ultra-0.5 centrifugal filter unit with ultracel-10 membrane (UFC501096, Milipore). For dot blot assay, 1 μl aliquots of 5 x concentrated samples were spotted on nitrocellulose membranes (Protean BA 85, GE Healthcare Life Sciences, Whatman). Released proteins were denatured, separated with 4–20% SDS-PAGE (Criterion TGX, Bio-Rad) and transferred to an Immuno-Blot TM polyvinylidene difluoride (PVDF) membrane (Bio-Rad) in western blot analysis. After blocking the membrane in 3% BSA dissolved in TBS, membranes were incubated 2 h at room temperature in 3% BSA with a primary polyclonal antibody GRP78 supplied with the GRP78-ELISA Kit (Biovendor). Afterwards, membranes were incubated with an anti-biotin HRP conjugated antibody (CST) diluted 1:1000. After washing, a chemiluminescent substrate (LumiGLO Reagent and Peroxide, Cell Signaling Technology) was added to the membrane, which was then exposed in the Chemidoc XRS station (Bio-Rad Laboratories).

Twenty μg of protein extract were denatured, separated by a 4-15% SDS-PAGE (Criterion TGX, Bio-Rad) and transferred to nitrocellulose membrane (Whatman). After blocking the membrane in 5% non-fat milk powder dissolved in PBS with 0.1% Tween, membranes were incubated in 0.5% non-fat milk powder at 4°C overnight with 0.1 μg/mL final concentration of detection antibody (BAF960), 0.1 μg/mL final concentration of capture antibody (MAB9601) or 0.1 μg/mL final concentration of mouse anti-human EpCAM (clone C-10, SCBT). Afterwards, membranes were incubated with a HRP-conjugated rabbit anti-mouse IgG (Dako Cytomation) for capture antibody and a HRP-conjugated rabbit anti-goat IgG (Dako Cytomation) for detection antibody, respectively. Next, a dilution of 1:1,000 for 1 hour at room temperature was prepared. After washing, a chemoluminescent substrate (LumiGLO Reagent and Peroxide, Cell Signaling Technology) was added to the membranes and protein was detected in the Chemidoc XRS station (Biorad Laboratories).
For Dot Blot analysis 10 ng of FLAG-tagged EpCAM produced in HEK293FT cells dissolved in assay buffer (1%BSA/PBS) or serum (n = 6, healthy probands) or ascites (n = 12) were spotted onto nitrocellulose membrane.

To verify the production of PilA-FLAG proteins, cell lysates were prepared as described previously with minor modifications (77 (link)). Briefly, after overnight growth, the bacterial culture was back diluted 1:100 in 2 ml of LB and grown at 37°C for the indicated amount of time (for time course experiments) or up to an OD₆₀₀ of ∼0.65. At the time of harvesting, the bacteria were centrifuged for 3 min, and the pelleted cells were resuspended in 2× Laemmli buffer, whereby the volume was adjusted according to the total number of bacteria (100 μl buffer per OD₆₀₀ unit). The resuspended samples were incubated at 95°C for 15 min. Proteins were separated by SDS-PAGE using 15% resolving gels and blotted onto\polyvinylidene difluoride (PVDF) membranes as previously described (76 (link)). Primary monoclonal antibodies against the FLAG tag (ANTI-FLAG M2; Sigma-Aldrich) were used at 1:2,000 dilution, and goat anti-mouse antibody—horseradish peroxidase (HRP) served as the secondary antibody (diluted 1:5,000; Sigma-Aldrich). Sigma70 was detected as a loading control using Direct-Blot anti-E. coli Sigma70-HRP-conjugated antibodies at a dilution of 1:10,000 (BioLegend, USA distributed via Brunschwig, Switzerland). Lumi-Light^PLUS Western blotting substrate (Roche) served as the HRP substrate. Luminescent signals were detected using a ChemiDoc XRS+ station (Bio-Rad).