Pchk1

Western blotting was performed as previously described (Zong et al, 2015 (link)). Briefly, cells were washed in PBS and lysed in an extraction buffer containing 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 5% Tween-20, 2% Igepal, 2 mM PMSF, 2.5 mM b-glycerophosphate (all from Sigma) and supplemented with protease and phosphatase inhibitor tablets (both Roche Diagnostics). Equal amounts of protein were loaded into precast 4–12% Bis-Tris mini-gels (Invitrogen) and resolved by SDS-PAGE. Thereafter, proteins were transferred onto nitrocellulose membranes, blocked in a TBS-based blocking agent (LiCor Biosciences) and incubated with the corresponding primary antibodies recognizing the following proteins: WRN (1:1000, Novus), FLAG (1:1000, Sigma), pKAP1 (S824, 1:1000, Bethyl), pATR (T1989, 1:500, Cell Signaling), pCHK1 (S345, 1:500, Cell Signaling), pCHK1 (S317, 1:500, Novus), pCHK1 (S296, 1:1000, gift of Dr. Lee Jung-Min), CHK1 (1:1000, Cell Signaling), pRPA2 (S33, 1:1000, Bethyl), RPA2 (1:2000, Cell Signaling), pCHK2 (T68, 1:1000, Cell Signaling), CHK2 (1:1000, Cell Signaling), β-actin (1:5000, Sigma) and Tubulin (1:5000, Sigma). Fluorescently labeled secondary antibodies were used at a dilution of 1:10,000 (Li-Cor Biosciences). Visualization and quantification of protein bands was achieved by fluorescence imaging using a Li-Cor Odyssey CLx imaging system (Li-Cor Biosciences).

Sections (2 μm) of livers (fixed in 4% paraformaldehyde and paraffin-embedded) were stained with Hematoxylin/Eosin or various antibodies. Incubation in Ventana buffer and staining was performed on a NEXES immunohistochemistry robot (Ventana Instruments) using an IVIEW DAB Detection Kit (Ventana) or on a Bond MAX (Leica). Immunostainings were performed as described before (Wolf et al., 2014 (link)) with antibodies against the following proteins: Ki67, 1:200 dilution (SP6, NeoMarkers / Lab Vision Corporation); γH2AX, 1:300 dilution (Novus Biologicals); p-cJUN, 1:100 (Abcam); cleaved-Caspase8, 1:500; p-CHK1, 1:50 and p-CHK2, 1:500 (Novus Biologicals). For virtual microscopy and archiving, histological and immunohistochemical images were digitalized using a Nano Zoomer C9600 Virtual Slide Light microscope scanner by Hamamatsu using NDP, View Software, version 1.2.36. Alternatively, for quantification of stainings, slides were scanned using a SCN 400 slide scanner (Leica) and analyzed using Tissue IA image analysis software (Slidepath, Leica).