0.45 m nitrocellulose membrane

Protein isolation was performed by lysis of the cells in RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA), supplemented with complete protease and phosphatase inhibitors (Roche, Basel, Switzerland). Proteins were resolved on a 7.5% Bis-Tris gel and blotted onto a 0.45 µm nitrocellulose membrane (GE Healthcare Life Sciences, Chalfont St. Giles, UK). After blocking the membranes with 5% skimmed milk in Tris-buffered Saline Tween (TBS-T), they were incubated overnight with the respective primary antibodies (Supplementary Table S1). Membranes were washed 3× for 10 min with TBS-T. Secondary antibody incubation was performed for 1 h at RT, and membranes were subsequently washed 3× for 10 min with TBS-T. Amersham ECL Prime Western Blotting Detection Reagent was used for the chemiluminescent detection (GE Healthcare Life Sciences) and captured with the imaging device Fusion FX.

Cells were grown to 80% confluence in a six-well plate and lysed with radioimmunoprecipitation assay buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1% Triton X-100, and 0.1% SDS) supplemented with protease and phosphatase inhibitor cocktails (#78438, #78427; Thermo Fisher Scientific). Lysates were spun at maximum speed for 10 min before protein levels were measured using Precision Red (#ADV02; Cytoskeleton). Samples were run using precast 4–12% NuPAGE Bis-Tris Acrylamide Gels (#NP0321; Thermo Fisher Scientific) and transferred to a 0.45-µm nitrocellulose membrane (#10600002; GE Healthcare). Membranes were blocked in 5% BSA in TBS + 0.1% Tween-20 for 30 min before being incubated with specific primary antibodies overnight and the corresponding fluorescently conjugated secondary antibody for 1 h (#A21206 and #A10038, Invitrogen; #SA5-35521 and #SA5-35571, Thermo Fisher Scientific) before being analyzed using Image Studio Lite (LiCor).

For the Dot blot assay, fractions generated from both the sucrose and iodixanol gradients were subjected to lysis using RIPA buffer (150 mM NaCl, 1% IGEPAL, 0.5% sodium deoxycholate, 50 mM Tris, pH 8.0) with a ratio of 1:10 (buffer: sample) and incubated at 4°C for 30 min. After this process, 50 µL of the samples were applied onto a 0.45 µm nitrocellulose membrane (GE Healthcare, NJ, United States). The membrane was then incubated in a 3% (W/V) skimmed milk blocking solution for 1 h, washed three times with PBS for 10 min, and incubated under agitation with the Mab 4G2 (1:100) or the anti-gp64-baculovirus (Mab gp64) monoclonal antibody (1:2000) for 90 min. The membranes were then washed three times each with PBS for 10 min and incubated under agitation with HRP-conjugated goat anti-mouse secondary antibody (1:2000) for 1 h. The membrane was developed with the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, IL, United States) on the photo-documenter Uvitec Cambridge, with analysis in the Alliance program (Uvitec Cambridge, CB, United Kingdom) For the Western blot assay, the same methodology described for detection of CprME -ZIKV protein expression in infected Sf9 cells was followed.

Secreted protein fractions and corresponding whole-cell lysates (obtained by boiling the cell pellet in sample buffer as above) were used for immunoblot analysis. Samples were separated by SDS-PAGE and were transferred from a 4–12% Bis-Tris NuPAGE gel to a 0.45 µM nitrocellulose membrane (GE Healthcare) using an XCell II Blot module (Invitrogen) at 30 V for 90 min. Membranes were then blocked with 5% skim milk powder in PBS-Tween at room temperature for one hour before being incubated with primary antibodies for one hour. Membranes were washed three times in PBS-Tween for 10 min each before incubating for one hour with secondary antibodies. Primary antibodies used were anti-EspD (1:2500) and anti-GroEL (1:25,000). Secondary antibodies used were anti-mouse and anti-rabbit HRP-conjugated (1:20,000). Immunoblots were incubated with SuperSignal West Pico chemiluminescent substrate (Pierce) for five minutes before imaging using a G:Box Chemi system (Syngene).

All cultivation analyses were performed immediately after sampling to minimize preselection bias during transport and storage. Different selective agar systems were used for the detection of E. coli (Chromocult-Coliform-Agar, Merck, Darmstadt), Acinetobacter spp. (Acinetobacter-CHROMagar, MAST Diagnostica GmbH, Reinfeld, Germany), intestinal enterococci (Slanetz-Barley Oxoid, VWR, Darmstadt and Bile esculin Agar, VWR, Darmstadt), and extended spectrum beta lactamase (ESBL) producing Gram-negative bacteria (CHROMagar^TM MAST Diagnostica GmbH, Reinfeld). Three different sample volumes (750 mL, 1000 mL, and 1250 mL) were filtered through a 0.45 µm nitrocellulose membrane (GE Healthcare Life sciences, Solingen, Germany). The membrane was then transferred onto an ager plate and incubated under conditions recommended by the manufacturer. After an appropriate incubation time, Colony Forming Units (CFU) were determined for each plate and calculated as CFU per 100 mL. The mean of all three sample volumes were calculated for final evaluation.

Protein isolation was performed via lysis of the cells in RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) supplemented with complete protease and phosphatase inhibitors (Roche, Basel, Switzerland). The proteins were separated on a 7.5% Bis-Tris gel and blotted onto a 0.45 µm nitrocellulose membrane (GE Healthcare Life Sciences, Chalfont St. Giles, UK). After blocking the membranes with 5% skimmed milk in Tris-buffered saline tween (TBS-T), they were incubated overnight with the respective primary antibodies (Supplementary Table S1). The membranes were washed 3× for 10 min with TBS-T. Secondary antibody incubation was performed for 1 h at RT and the membranes were subsequently washed 3× for 10 min with TBS-T. Amersham ECL Prime Western Blotting Detection Reagent was used for the chemiluminescent detection (GE Healthcare Life Sciences) and captured with the imaging device Fusion FX.

For protein extraction, cells were harvested and lysed in RIPA buffer (Sigma-Aldrich Chemie GmbH, Germany) supplemented with complete protease and phosphatase inhibitors cocktail (Roche, Switzerland). The lysates were separated on a 4–20% Bis-Tris gel and blotted onto a 0.45 µm nitrocellulose membrane (GE Healthcare Life Sciences, Germany). The membranes were then blocked with 5% skimmed milk in Tris-Buffered Saline Tween (TBS-T) and incubated overnight with the respective primary antibodies: Total Smad 1 (1:1000, TBS-T 5% BSA; CST, USA), phospho Smad 1/5/8 (1:1000, TBS-T 5% milk; CST, USA), Total Smad 2/3 (CST, 1:1000, TBS-T 5% BSA), and phospho Smad 2/3 (1:1000, TBS-T 5% milk; CST, USA). After incubation with the appropriate secondary antibodies, signals were acquired with a Fusion-FX7 imaging system.