Transfer buffer

The platelets of one healthy individual and VWF-deficient patients (negative control) were washed twice in PBS and clarified by centrifugation at 2500 rpm for 5 min. The cell pellets were re-suspended in a lysis mixture (50 mM TRIS-HCl, pH 8.0, 120 mM NaCl, 0,5% Nonidet P-40, 100 mM phenylmethylsulfonylfluoride, 1 µg ml⁻¹ aprotinin) at 4°C for 30 min. The lysate was clarified by centrifugation at 14000 rpm for 15 min. The protein samples were mixed 1∶1 with Laemmli buffer (190 µl Laemmli, 10 µl DTT) and denatured at 95°C for 5 min and cooled on ice. The protein lysates were separated using SDS-PAGE. After equilibration with transfer buffer (BioRad, Hercules, California), the proteins were transferred from the gel onto nitrocellulose membranes using a semi-dry sandwich technique (0.1 A, 2 h). After blocking with human milk powder (100 ml TBS, 5 g human milk powder, 200 µl NaN₃ at room temperature overnight), the membranes were incubated with primary antibodies (ATZ11 (1∶50), anti-VWF (1∶400)) overnight. After incubation with the goat anti-mouse/rabbit-conjugated secondary antibody for 2 h, the membranes were stained with an alkaline phosphatase conjugate kit (BioRad, Munich, Germany) and 5-bromo-4-chloro-3-indolylphosphate as the chromogen.

Total bacterial proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes in transfer buffer (Bio-Rad, 1X Tris/Glycine Buffer) at RT as previously described (Zhang et al., 2017 (link)). Briefly, PVDF membranes were blocked in 5% (w/v) nonfat milk in phosphate-buffed saline with 0.1 % (v/v) Tween 20 (PBST) for 2 h at RT prior to incubation with polyclonal rabbit antibodies (1:1,000 dilution) against the target protein at 4°C for 12 h. After washing with PBST, the membranes were incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit antibody (ComWin, Beijing, China) (1:5,000 dilution). The immunostained proteins were visualized using Clarity™ Western ECL Substrate (Bio-Rad, Hercules, CA, USA), and images were acquired with the ChemiDoc MP imaging system with Image Lab software (Bio-Rad). Coomassie R-350 was used to stain the PVDF membranes after blotting as a loading control.

Cell lines, tumors from cell-line xenografts, and tumors from patient-derived xenografts (PDX) were prepared and lysed in lysis buffer (20 mmol/L Tris, 150 mmol/L NaCI, 1% NP-40, 1 mmol/L EDTA, 1 mmol/L EGTA, 10% glycerol, and protease and phosphatase inhibitors). The samples were incubated on ice for 30 minutes, then centrifuged at 14,000 rpm for 10 minutes at 4°C. Tumor lysates were homogenized with Tissuemiser (Thermo Fisher Scientific) in the lysis buffer described previously, incubated for 30 minutes on ice, and centrifuged at 14,000 rpm for 10 minutes at 4°C. Protein concentration was determined by BCA Protein Assay (Pierce). Proteins were resolved using the NuPAGE Novex Midi Gel system on 4% to 12% Bis–Tris gels (Invitrogen), transferred to polyvinylidene difluoride membranes (PerkinElmer) in transfer buffer (Bio-Rad) with 20% methanol. Following transferring, the membrane was blocked in PBS-T with 5% nonfat milk for 1 hour and then incubated with the indicated antibodies overnight. After secondary antibody (GE Healthcare) incubation, the antibodies on the membranes were detected with the Syngene G: Box camera (Synoptics). Representative blots are shown in the figures.