Gapdh hrp

For western blotting, samples from normal livers and tumors excised from livers were homogenized with calculated volume of 2× sample buffer (1 M TRIS/HCl, 10% SDS, 0.1% bromophenol-blue, 10% β−mercaptoethanol, 10% glycerol) and heated for 15 min at 95 °C. Proteins were resolved on protein gels (Bio-Rad, Cat#4568083) and transferred onto PVDF membrane (Bio-Rad, Cat#170-4272) with Trans-blot Turbo transfer system (Bio-Rad, Cat#1704150). After being incubated with the primary antibody, horseradish peroxidase-(HRP) conjugated secondary antibody (Novex, Life technologies) at 1: 5000 was used for 1 h incubation. The signals were detected by chemiluminescence (ECL, Thermo scientific). Images were taken with Li-COR Odyssey FC (Li-COR, Cat#2800). C-MYC (Cell Signaling Technology, 5605 S, RRID:AB_1903938, 1:1000), PRKDC (DNA-PK))Novus, sc57-08, RRID: AB_2809479, 1:1000), β-actin (Sigma, A5441, RRID:AB_476744, 1:5000) and GAPDH-HRP (Cell Signaling Technology, 3683 S, RRID:AB_1642205, 1:1000) were used for western blot.

Proteins were extracted in radioimmunoprecipitation buffer and quantified as described in (45 (link)). A total of 10 μg of protein was loaded in each well of a gel (Bio-Rad # 567-1094, 567-1095, 456-1036). Blots were blocked in 5% milk/tris buffered saline with tween (TBST) for 20 min to 2 h at room temperature (RT) or overnight at 4 °C. Primary antibodies to Nanog (Millipore; Cat # 5731) was used at 1:1000, beta-Actin (Sigma; Cat # a5441) was used at 1:5000 in 5% milk/TBST or GAPDH-HRP (Cell Signaling Tech; 51332S) at 1:3000 in 5% milk/TBST for 90 min at RT or overnight at 4 °C. Blots were then washed with TBST and secondary antibody donkey anti-rabbit IgG-HRP (Santa Cruz; Cat # sc2313) was used at 1:5000 for 30 to 60 min at RT for Nanog. For beta-Actin, a secondary antibody (Santa Cruz; Cat # sc2064) goat anti-mouse IgM-HRP was used at 1:5000 for 30 min at RT. Blots were washed with TBST and then antibody labeled proteins were detected using Amersham ECL Prime Western Blotting Detection Reagent (Cat # RPN2232).

All proteins were resolved by SDS-PAGE and transferred to a PVDF membrane (PerkinElmer). Membranes were blocked in TBS-T (50 mM Tris, pH 7.5, 0.1% Tween-20, 150 mM NaCl) containing 5% milk. Following blocking, primary antibodies were added in block solution overnight at 4 °C on a rocker. Multiple antibodies were used for some original blots shown in Additional File 3: Figure S1 if there could be at least two ladder markers still be present around each protein of interest. For blots that were trimmed, images from all replicates are included. The next day, all blots were washed three times in TBS-T for 5 min each wash and secondary antibodies were added for 1 h at room temperature. Following an additional three washes for 5 min each, images were obtained on a Bio-Rad ChemiDoc MP instrument using the Clarity ECL substrate. Antibodies used for immunoblotting were as follows: PDK1 (used for PDPK1, Cell Signaling, 13,037), N-MYC (Cell Signaling, 51,705), WDR5 (Cell Signaling, 13,105), Flag-HRP (Cell Signaling, 86,861), GAPDH-HRP (Cell Signaling, D16H11).

To isolate protein from whole-cell lysis for immunoblotting, cells were lysed in RIPA buffer (10 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.1% SDS) supplemented with protease inhibitor cocktail (Roche). Protein samples were separated by SDS-PAGE, transferred onto nitrocellulose membrane and blocked with 5% milk. Membranes were incubated with primary antibody overnight at 4 °C and secondary antibody for 1 h at room temperature. Immunoblot membranes were developed with chemiluminescent substrate (Thermo Fisher Scientific) and imaged using Chemidoc XRS+ with Image Lab 6.0.1 software (BioRad, Hercules, CA). Densitometry quantification was performed using Image J, version 1.53e. Primary antibodies used were: anti-total DDR1 (5583; Cell Signaling Technology, clone D1G6, 1:1000), GAPDH-HRP (3683, Cell Signaling Technology, clone 14C10, 1:1000), anti-cleaved NOTCH1 (4147, Cell Signaling Technology, clone D3B8, 1:1000), anti-phospho-DDR1 Y513 (14531, Cell Signaling Technology, clone E1N8F, 1:1000), anti-JAG1 (2620, Cell Signaling Technology, clone 28H8, 1:1000) and anti-β-Tubulin- HRP (5346; Cell Signaling Technology, clone 9F3, 1:1000). Secondary antibody used was anti-rabbit-HRP (7074, Cell Signaling, 1:5000).