Las 3000 mini imaging system

Different fractions, obtained during purification of recombinant AtRS4, were separated and analyzed for purity by SDS-PAGE using a 10% acrylamide separation gel and colloidal Coomassie blue staining. For Western blots, unstained gels were blotted onto a nitrocellulose membrane (Schleicher and Schuell Protran^TM BA85) for 1 h at 100 V. Non-specific binding sites were blocked by incubating the membrane for 1 h with TBST-BSA (1% w/v) at room temperature followed by two washing steps with TBST each for 15 min. For detection of His-tagged protein, a SuperSignal West HisProbe kit (Thermo Scientific, Vienna, Austria) was used. Luminescence detection was performed using an LAS 3000 mini imaging system (Fujifilm, Dusseldorf, Germany).

Proteins were extracted using with RIPA buffer with 1% Protease inhibitor cocktail (Nacalai Tesque). After determination of the protein concentration using Protein Quantification Assay (Takara Bio Inc.), all samples were denatured in Laemmli sample buffer for 5 min at 100 °C. The denatured samples were separated by SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (EMD Millipore). After blocking with 5% milk or 5% BSA, the membranes were incubated with the primary antibodies as follows: β-actin (1:1000, Sigma, cat#: A5316), BRD2 (1:1000, Abcam, cat#: ab139690), BRD3 (1:1000, Santa cruz, cat#: sc-81202), BRD4 (1:1000, Cell signaling, cat#: 13440), BRD4 (1:1000, abcam, cat#: 128874). Cleaved caspase3 (1:1000, Cell signaling, cat#: 9664), LaminB1 (1:1000, Abcam, cat#: ab16048), p21 (1:1000, Cell signaling, cat#: 2947 or Abcam, cat#: ab107099), XRCC4 (1:1000, Santa cruz, cat#: sc-2711087). The membranes were then incubated with the secondary antibodies (1:1000, Cell signaling) and visualized with Amersham ECL prime/select (GE Healthcare), followed by detection with chemiluminescence using LAS-3000mini imaging system (Fujifilm) and by anlaysis of data using Multi Guage V3.1 (Fujifilm). Uncropped and unprocessed scans of the blots are included in the Source Data file.

Microsomal fractions were prepared as described previously [25 (link)] and stored at − 70°C until required. Microsomal protein concentrations were determined using the Bio-Rad Protein Assay Reagent (Bio-Rad Laboratories) and Western blot analysis was preformed as described previously [26 (link)] using polyclonal antisera to rat P450s and POR [27 (link),28 (link)]. Immunoreactive proteins were detected using horseradish peroxidase (HRP)-conjugated polyclonal goat anti-rabbit, anti-mouse or anti-sheep immunoglobulin secondary antibodies, as appropriate (Dako). Bands were visualized using Immobilon™ chemiluminescent HRP-conjugated substrate (Millipore) and a FUJIFILM LAS-3000 mini imaging system (Fujifilm UK.). Densitometric analysis was performed using Multi Gauge V2.2 software (Fujifilm UK).

Each sample was subjected to 7.5%, 12.5% or 15% (v/v) SDS-PAGE using a Tris-glycine buffer system, and the separated proteins were transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 3% (v/v) skim milk and 1% (v/v) bovine serum albumin (BSA) in PBS containing 0.1% (w/v) Tween-20 for 3 hours, followed by incubation at 4 °C overnight with the indicated primary antibody at an appropriate dilution (1:2,000) in PBS containing 3% (v/v) skim milk, 1% (v/v) BSA, and 0.1% (w/v) Tween-20. The membranes were washed and then incubated with a horseradish peroxidase-conjugated secondary antibody (1:2,000) (Cell Signaling Technology, Inc., Danvers, MA, USA) at room temperature for 1 hour. Signals were detected using Pierce Western Blotting Substrate Plus (Thermo Scientific, Southfield, MI, USA). The intensities of specific bands were quantitatively measured using an LAS-3000 mini imaging system (FujiFilm Co., Tokyo, Japan) equipped with Multi-Gauge Ver3.0 software (FujiFilm). As an internal control, the expression of β-actin was measured.