Fuji image analyzer las 1000uvmini

Southern blotting was performed as described previously [23 (link)]. Briefly, 30 μg of genomic DNA was digested with EcoRV and StuI at 37°C and electrophoresed on a 0.8% agarose gel at 15 V for 14–18 h. Subsequently, DNA fragments were transferred to an Immobilon-Ny⁺ Transfer Membrane (Millipore, Concord Road, Billerica, MA, USA) by an alkaline transfer technique, followed by hybridization for >18 h at 55°C in hybridization buffer. The probe used was PCR amplified with primers HPRT 3′probe Fw and HPRT 3′probe Rv (Additional file 1: Table S1). Southern hybridization was performed using Amersham Gene Images AlkPhos Direct Labelling and Detection System (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). Signals were detected with CDP-Star (Roche, Basel, Switzerland), and analyzed by using the Fuji Image Analyzer LAS-1000UVmini (Fuji Film Co., Tokyo, Japan).

Cells were rinsed twice with PBS⁻ and scraped in 200 μl of lysis buffer (50 mM Tris-HCl (pH 6.8), 2% sodium dodecylsulfate, 10% glycerol, 100 μM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) containing Protease Inhibitor Cocktail (1/10 volume of buffer; Sigma-Aldrich). The lysates were allowed to stand for 20 min at 4 °C and, after sonication, centrifuged for 30 min at 16,000 g. The supernatants were collected and used for western blot analysis. Twenty micrograms of the lysates were electrophoresed in a 7.5% polyacrylamide gel and then transferred onto a polyvinylidene difluoride membrane (Millipore). The membrane was probed with anti-Lig4 antibody (1:500, kindly provided by Dr Hirobumi Teraoka)37 (link) or anti-Actin antibody (1:2,000, A2066, Sigma-Aldrich). Signals were detected with Clarity Western ECL Substrate (Bio-Rad) and analysed using a Fuji Image Analyzer LAS-1000UVmini (Fuji Film Co.). Full scans of the blots are shown in Supplementary Fig. 2.