Eblot protein transfer device

Western blotting was performed as described previously (Ballesteros-Plaza et al., 2013 (link)) with some modifications: E. coli cells were sonicated. E. coli cell (1 × 10⁸ CFU) lysates were boiled in 5× loading buffer [0.25M Tris-HCL (pH 6.8); 10% (w/v) sodium dodecyl sulfate; 0.5% (w/v) bromophenol blue; 50% (v/v) glycerol; 5% (w/v) β-mercaptoethanol], and proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes using eBlot Protein Transfer Device (Genscript), according to manufacturer’s instructions. Membranes were probed with anti-E. coli AmpC Ab (mouse polyclonal antiserum; for details of preparation, see Supplementary Data) and anti-GAPDH Ab (1:1000 in PBS, rabbit polyclonal, #E-AB-20059, from Elabscience) followed by horseradish peroxidase (HRP)-conjugated goat anti-mouse Antibody (1:5000 in PBS, #bsm-0294M-HRP, Bioss, China). Chemiluminescence was developed using an Immobilon Western chemiluminescent HRP substrate (Millipore) and documented using a Tanon 5200 milti (Biotanon). Densitometry was performed using ImageJ software (Ver 1.4.3.67) and was normalized to GapA (probed by anti-GAPDH Ab) (Wu et al., 2012 (link)).

Protein extraction was performed by washing the cell pellet with ice-cold PBS. Next, 1 mL of ice-cold RIPA lysis buffer (Sigma) and a protease inhibitor cocktail (Thermo Scientific, Rockford, IL, USA) were added and incubated on ice for 30 min. Then, 30 µg of cell lysates were resolved on 4–20% ExpressPluS PAGE Gel (GenScript, Piscataway, NJ, USA) following concentration measurements. The eBlot Protein Transfer device (GenScript, Piscataway, NJ, USA) was used to transfer the proteins onto a PDVDF Transfer Membrane (Thermo Scientific, Rockford, IL, USA). The membranes were washed and incubated for 1 h with a secondary anti-mouse antibody conjugated with HRP (Cell Signaling Technology, Danvers, MA, USA). Pierce ECL Western blotting Substrate (Thermo Scientific, Rockford, IL, USA) and BioRad Universal Hood II with a Chemiluminescence System (BioRad, Hercules, CA, USA) were used to visualize the result. Antibodies: Recombinant Anti-FANCD2 antibody [EPR2302] (abcam—ab108928) 1/1000; Recombinant Anti-Rad51D antibody [EPR16205] (abcam—ab202063) 1/1000; Recombinant Anti-β Actin antibody [EPR21241] (abcam—ab213262) 1 µg/mL; Recombinant Anti-Rad51D antibody [EPR16205] (ab202063) 1/1000.

Protein extraction was performed incubating cell pellet with the mixture of RIPA buffer (Sigma) and protease inhibitor cocktail (Thermo Scientific, Rockford, Illinois, USA). After concentration measurement, 30 μg of cell lysates was resolved on 4–20% ExpressPluS PAGE Gel (GenScript, Piscataway, New Jersey, USA). The proteins were then transferred onto PVDF Transfer Membrane (Thermo Scientific, Rockford, Illinois, USA) using eBlot Protein Transfer device (GenScript, Piscataway, New Jersey, USA). Membranes were blocked and blotted overnight with primary antibodies recognizing LIG4 and GAPDH (Santa Cruz Biotechnology, Dallas, Texas, USA). Membranes were then washed and incubated 1 h with secondary anti-mouse antibody conjugated with HRP (Cell Signaling Technology, Danvers, Massachusetts, USA). The result was visualized using Pierce ECL Western Blotting Substrate (Thermo Scientific, Rockford, Illinois, USA) and BioRad Universal Hood II with Chemiluminescence System (BioRad, Hercules, California, USA).