Sample loading buffer

All cell extracts were harvested in 1X RIPA buffer (Cell Signaling Technology, Inc.; Danvers, MA), and samples were centrifuged at 12,000g at 4°C for 25 min. The samples were then boiled in sample loading buffer (Invitrogen) containing SDS, and equal amounts of samples were resolved on a 4–12% SDS-PAGE gradient gel and then transferred onto a nitrocellulose membrane. Unbound sites on the membrane were blocked, and the membrane was incubated with the indicated antibodies overnight at 4°C. We used anti-CYP3A4, anti-MDR1, anti-PXR, anti-FLAG M2 (1:1000 dilutions) and anti-β-actin (1:5000 dilution) antibodies to detect CYP3A4, MDR1, PXR, FLAG-PXR and β-actin, respectively. All Western blot analyses were performed on the Odyssey Infrared Imaging system (LI-COR Biosciences; Lincoln, NE). The intensity of each protein band was quantified using ImageJ 1.48 software [35 (link)]. The intensity of each protein band was normalized to that of actin to generate the relative intensity, with the relative intensity of the DMSO treated sample set as “1”.

NK cells were isolated from C57BL/6 mouse spleens with an NK cell negative selection isolation kit (Miltenyi biotec, Bergisch Gladbach). Cells were suspended in 1× Cell Lysis Buffer containing protease and phosphatase inhibitors. Lysate was diluted to 1 μg/μL in lysis buffer, and 100 μL of the diluted lysate was incubated with anti-CD122 antibody (Santa Cruz Biotechnologies, Dallas TX) at a 1:100 dilution overnight at 4°C with end-over-end rotation. Magnetic protein G beads (Bio-Rad) were washed three times with 1× PBS, added to lysate–antibody complexes, and the mixture was incubated overnight at 4°C with end-over-end rotation. Beads were washed three times with cold 1:1 lysis buffer:PBS, removing supernatant after each wash. Protein was eluted from beads by adding 1× sample loading buffer (Invitrogen) containing reducing agent (Invitrogen), and boiling beads at 95°C for 15 minutes. A total of 100 μg equivalent starting material was resolved on a 10% SDS-PAGE gel, and then transferred to Immuno-Blot PVDF membrane (Bio-Rad). Membranes were probed for total phosphotyrosine using anti-phosphotyrosin 4G10 (Cell Signaling Technology).

HEK293 cells irradiated (4 Gy) or not irradiated were collected 10 min
post IR and washed in 1xTBS (ice-cold) and resuspended in 0.5mL of lysis buffer
[50mM Tris, pH7.5; 150mM NaCl; 0.5% NP-40; 5mM MgCl2; 5% glycerol; 1X Protease
inhibitors (Roche tablet); 1X phosphatase inhibitors tablet (Roche)]
supplemented with 1μL per 1mL Benzonase (250 units/mL, Sigma). Lysates
were incubated at 4°C for 45 min. Lysates were cleared and equal amount
of total protein extracts were used for each sample and primary antibody,
pre-incubated with G dynabeads (Invitrogen), was added and left at 4°C on
a wheel for further 2h. The beads were gently collected using a magnet rack
(Invitrogen) and washed 6 times with 1X lysis buffer and resuspended in
50μL of sample loading buffer (Invitrogen).

All cell extracts were harvested in 1× RIPA buffer from Cell Signaling Technology, Inc. (Danvers, MA), and samples were centrifuged at 12,000 × g at 4 °C for 25 min. The samples were then boiled in sample loading buffer (Invitrogen) containing SDS, and equal amounts of samples were resolved on 4–12% SDS-PAGE gradient gels and then transferred onto nitrocellulose membrane. The membrane was blocked and incubated with the indicated antibodies overnight at 4 °C. All Western blot analyses were performed on the Odyssey Infrared Imaging system (LI-COR Biosciences, Lincoln, NE). The intensity of each protein band was quantified using ImageJ 1.48 software [25 (link)]. The intensity of each protein band was normalized to that of β-actin to generate the relative intensity, with the relative intensity of the WT hPXR sample set as “1”.