G box imager

Samples were lysed in Mammalian Cell Lysis Buffer (50 mM Tris, pH 8.0, 2 mM dithiothreitol (DTT), 5 mM ethylenediaminetetraacetic acid (EDTA), 0.5 % NP-40, 100 mM NaCl) with added protease inhibitors (1 μM mycrocystin, 2 mM phenylmethanesulfonyl fluoride (PMSF), 1 × protease inhibitor cocktail (Sigma-Aldrich; P8340-5ML), 1 × phosphatase inhibitor cocktail (Calbiochem, San Diego, CA, USA; 524625-1SET)), and 10–30 μg total protein was loaded onto Bio-Rad (Hercules, CA USA) Criterion gels and transferred to nitrocellulose for Western blotting using standard procedures. Antibodies used for Western blotting included those recognizing BTG2 (sc-33775, Santa Cruz Biotechnology Inc., Dallas, TX, USA), and α-tubulin (2144, Cell Signaling Technologies). Protein was detected using enhanced chemiluminesence (ECL) (34080, Thermo Fisher Scientific) on a G-Box imager (Syngene, Frederick, MD, USA).

Purified FliW-His₆ protein in 50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 0.01% Tween-20 was incubated with 10 μl of Dynabeads^TM His-Tag Isolation & Pulldown (Invitrogen) for 10 min, and the beads were subsequently washed four times with the same buffer. E. coli BL21(DE3)pLysS cells expressing wild type CsrA or CsrA proteins containing C-terminal deletions were resuspended in 20 mM sodium phosphate, pH 7.5, 150 mM NaCl, 0.01% Tween-20, and lysed by sonication. Insoluble material was removed by centrifugation at 12,000 × g and lysates were then incubated with FliW-coated beads for 20 min at room temperature. Beads were subsequently washed four times with 300 μl of the same buffer. Protein complexes were eluted from magnetic beads with 50 mM sodium phosphate, pH 7.5, 150 mM NaCl, 300 mM imidazole.
For Western blot analysis, samples were separated on a 15% SDS-PAGE gel and then electroblotted onto PVDF membranes (Millipore). Membranes were probed overnight with 1:200 dilution of primary antibodies against CsrA in TBS-T (Tris-buffered saline, 0.05% Tween 20). After washing twice with TBS-T, horseradish peroxidase-conjugated secondary goat anti-rabbit antibodies (Bio-Rad, 1:20,000 in TBS-T) were applied. Blots were washed twice and incubated in SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific). Specific bands were visualized using G:Box imager (Syngene).

Following irradiation with different radiation qualities, cells were harvested 24 h after treatment and proteins extracted according to published methods (34 (link)). A 40- μ g total protein sample was loaded onto a 10% SDS-PAGE gel, and after electrophoresis, the proteins were blotted on a nitrocellulose membrane (Life Technologies, USA). The membranes were blocked with 5% non-fat dairy milk in PBS-Tween (0.1% Tween-20 in PBS) and incubated overnight at 4°C with primary antibody [PARP #9542 (Cell Signaling, USA) at a dilution of 1:1,000 in 5% non-fat milk in PBS]. The anti-β-actin (Cell Signaling, USA) antibody was used as a housekeeping control at a dilution of 1:5,000. After washing with PBS-Tween, the membranes were incubated in their secondary anti-rabbit and anti-mouse horseradish peroxidase-conjugated antibodies diluted at 1:2,000 at room temperature for 1 h. The membranes were then washed and developed with Luminata Crescendo Western HRP substrate (Millipore, USA) using the GBox Imager by Syngene (Cambridge, UK).