Western Blot Quantification Protocol

Bacteria were grown as described above. At OD₆₀₀ 0.5, 30 ml samples were taken for protein extraction as described by Menendez-Gil et al. (2020) (link). Western blotting was performed as previously described (Caballero et al., 2018 (link)). The 3xFLAG tagged protein samples were incubated with mouse monoclonal anti-FLAG M2-Peroxidase (HRP) antibodies (Sigma) diluted 1:1,000, whereas the GFP samples were incubated with mouse monoclonal anti-GFP antibodies 1:5,000 (Living Colors, Clontech) and peroxidase-conjugated goat anti-mouse immunoglobulin G and M antibodies 1:2,500 (Pierce-Thermo Scientific). Membranes were developed using the SuperSignal West Pico Chemiluminiscent Substrate kit (Thermo Scientific). Mean intensities of developed protein bands were quantified by densitometry of Western blot images using ImageJ and plotted as arbitrary units (A.U.). Statistical significances were calculated by running a paired t-test in GraphPad Prim; asterisks (*) indicate p-values lower than 0.05 (p < 0.05) while ns indicate not significant differences.