Monoclonal rabbit anti ha antibodies

The protein extractions and Western blot analyses were performed as previously described [20 (link)]. The membranes were incubated overnight at 4 °C with a 1:5000 dilution of monoclonal rabbit anti-HA antibodies (Sigma-Aldrich, Saint-Louis, MO, USA) or 1:5000 dilution of monoclonal rabbit anti-FLAG antibodies (Sigma-Aldrich, Saint-Louis, MO, USA).

Exponentially growing bacteria (15 mL, A_650nm = 0.3), grown at 30 °C, were exposed to 1 or 5 µg/mL mitomycin C. After 3 h at 30 °C with continuous shaking, cells were harvested by centrifugation at 4 °C and the pellets washed with 1X cold saline-sodium citrate (SSC) buffer. Then, the bacteria pellets were re-suspended in 150 µL of SSC 1X with 0.4 mM protease inhibitor cocktail (Roche) and cells disrupted with a FastPrep Instrument using 0.1 g of glass beads (500 µm) and four pulses of 30 s. Cell debris were removed by centrifugation at 20,000× g for 10 min at 4 °C, and the supernatant fluid collected and placed in sterile microcentrifuge tubes. Protein concentrations were determined by Bradford assay (Bio-Rad). Proteins were subjected to electrophoresis through a 12% Glycine SDS-PAGE gel (Mini-PROTEAN TGX Stain-Free Precast gel, Bio-Rad, Hercules, CA, USA) and transferred onto a polyvinylidene difluoride (PVDF) membrane (GE Healthcare, Chicago, IL, USA). All these experiments were performed three times as previously described [20 (link)] with a 15,000 dilution of anti-V5 rabbit primary antibody (Abcam, Cambridge, UK) or with a 1:5000 dilution of monoclonal rabbit anti-HA antibodies (Sigma-Aldrich, Budapest, Hungary).