Bicinchoninic acid (bca)

E.Coli DH5α bacteria as well as HEK cells were separately lysed in 100 mM HEPES pH 7.5 supplemented with 0.1% (v/v) SDS, 0.57 mM PMSF and 10 mM EDTA, heated for 10 min at 95 °C followed by ultrasonication (Bioruptor, 20 cycles, 45/15 s on/off time, high intensity) and centrifugation (13,000g, 5 min), thereupon only using the clear supernatant. Protein concentration of each supernatant was determined using BCA (Thermo Fisher Scientific, Waltham, USA) before subjecting 1 mg of each lysate to individual in-solution tryptic digestion as described above. Peptide desalting was performed using Sep-Pak C18 Plus Short Cartridge (Waters, Milford, USA) according to manufacturer´s instructions. After determining the peptide concentration via BCA (Thermo Fisher Scientific, Waltham, USA), 25 µg HEK eluate were mixed with 0.5 µg, 1.5 µg, 4.0 µg or without E. coli eluate to obtain peptide mass ratios of E.coli:HEK 1:50, 1:17, 1:6 and “HEK only”, respectively. For each mass ratio, four replicates were prepared resulting in 16 samples in total. Isobaric labelling with TMTpro-16plex and subsequent offline high pH fractionation was performed like described above before resulting fractions were vacuum dried and stored at − 80 °C until measurement.

Western blotting was conducted as described previously [39 (link)]. Lysed samples were measured using bicinchoninic acid (Thermo Scientific, Waltham, MA, USA) and bovine serum albumin was used as the standard for protein concentrations. Samples were prepared in a gel buffer [12.5 mM Tris buffer (pH 6.8), 4% sodium dodecyl sulfate (SDS), 20% glycerol, 10% 2-mercaptoethanol, and 0.2% bromophenol blue] and kept at 100 °C for 5 min. SDS-polyacrylamide gel electrophoresis containing with 8% to 14% acrylamide was used to separate equal concentrations of protein. Using a wet transfer system, the gels were transferred to polyvinylidene difluoride membranes at 90 V for 90 min. Membranes were immediately incubated in blocking buffer [10 mM Tris buffer (pH 7.5), 100 mM NaCl, 0.1% Tween 20, and 5% non-fat milk]. Blotting was done at 25 °C for 30 min and then membranes were incubated with specific primary antibody at 4 °C for 16 h. The secondary antibody was then added followed by an HRP conjugated anti-rabbit, anti-goat antibody, or anti-mouse antibody at 25 °C for 1 h. Antibody labeling was used to detect antibodies with WesternBright^TM ECL reagent (Advansta, Menlo Park, CA, USA).

Cells were washed in ice‐cold PBS and sedimented at 1000 g for 10 min at RT. Pellets were suspended in RIPA buffer (10 mm Tris/HCl, pH 7.4, 1% sodium deoxycholate, 1% Triton X‐100, 0.1% SDS) containing 1 mm PMSF and protease inhibitor cocktail (Sigma‐Aldrich). Protein concentration was quantified using Bicinchoninic acid (Thermo Scientific, Rockford, IL, USA). Total proteins were separated by SDS/PAGE and transferred to Porablot NCP membranes (Macherey‐Nagel, Düren, Germany). Blots were probed with anti‐FLAG (1 : 2000; Sigma‐Aldrich), anti‐E‐cadherin (1 : 2000; Cell Signaling Technology, Danvers, MA, USA), anti‐N‐cadherin (1 : 2000; Cell Signaling Technology), anti‐Snail (1 : 2000; Cell Signaling Technology), and β‐actin (1 : 2000; Santa Cruz Biotechnology, Dallas, TX, USA) antibodies. Primary antibody binding was detected with anti‐goat IgG‐HRP (1 : 2000; Santa Cruz Biotechnology), anti‐mouse IgG‐HRP (1 : 2000; Santa Cruz Biotechnology), or anti‐rabbit IgG‐HRP (1 : 2000; Santa Cruz Biotechnology). Membranes were revealed using the EZ‐ECL chemiluminescence kit (Biological Industries, Haemek, Israel) and the ChemiDoc Touch Gel Imaging System (Bio‐Rad, Hércules, CA, USA).