Medical x ray film

Cleared cell lysates (10–20 μg of protein) or immunoprecipitates were denatured by boiling for 5 min at 95 °C and resolved by SDS-PAGE. The resolved proteins were electrophoretically transferred onto Amersham Protran 0.45-μm nitrocellulose membranes (GE Healthcare Life Sciences). Membranes were blocked using 5% (w/v) non-fat milk in Tris-buffered saline (TBS) (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 0.1% (v/v) TWEEN-20 (hereafter called TBST) for 1 h at RT on a bench-top platform rocker. The membranes were subsequently incubated with the appropriate primary antibodies (as detailed above) diluted in 5% (w/v) milk-TBST overnight (~16 h) at 4 °C with continuous agitation. Following this, membranes were washed three times for 5 min using TBST prior to incubation with the relevant species-specific horseradish peroxidase (HRP)-conjugated secondary antibodies, also diluted in 5% (w/v) milk-TBST for 1 h at RT on a bench-top platform rocker. The membranes were subsequently washed three times for 5 min using TBST prior to enhanced chemiluminescence detection, and exposed onto Medical X-Ray Film (Konica Minolta) or Amersham Hyperfilm ECL (GE Healthcare Life Sciences) under safelight conditions. The films were developed using an SRX-101A automated medical film processor (Konica Minolta).

All expression trials and coimmunoprecipitation
samples were separated using NuPAGE 4–12% SDS-PAGE gels. The
proteins were then transferred to an Immobilon-P PVDF membrane using
an XCell II blot module. After transfer, membranes were stained with
PonceauS for 1 min to check the efficiency of protein transfer before
blocking with 10% skimmed milk at 4 °C for a minimum of 1 h.
Next, the membrane was incubated with anti-His-HRP (sc-8036 HRP, Santa
Cruz) for His-tagged RhoGDI-1 and RhoGDI-2, anti-FLAG-HRP (A8592,
Sigma-Aldrich) for FLAG-tagged RhoGDI-3, anti-V5-HRP (R961-25, ThermoFisher
Scientific) for all 23 small GTPases, anti-Rac1 (05-389, Merck), and
anti-GAPDH-HRP (ab9482, Abcam). Proteins were visualized by treating
with enhanced chemiluminescence solution for 2 min and exposing the
membrane to medical X-ray film (Konica).

Whole cell extracts were prepared from control or irradiated cells at the indicated time-points post irradiation by direct addition of 1 × Laemmli buffer to cells that still adhered to the culture plates following one wash with ice-cold PBS. Cells were disaggregated into the Laemmli buffer using a cell scraper, heated at 95°C for 5 min, sonicated and spun down at 14,000 g for 2 min at 4°C prior to quantifying protein content by the Bradford method. 30–50 μg of total cell extracts were separated using SDS-PAGE gels and transferred to nitrocellulose membranes. Chemiluminescence was detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and medical x-ray film (Konica Minolta Medical & Graphic Imaging Inc.,). In assays in which protein quantification was necessary, this was performed using a LiCor Odissey infrared imaging system according to manufacturer's instructions.