Fluorchem imager

For total protein, HHL-5 cells were washed twice with ice-cold PBS, harvested by scraping in 20 mM Tris-HCl (pH 8), 150 mM NaCl, 2 mM EDTA, 10% glycerol, 1% Nonidet P40 (NP-40) containing mini-complete proteinase inhibitor and 1 mM PMSF and then incubated in an ice bath for 20 min and centrifuged at 12,000 g for 15 min at 4°C. Supernatant was collected and the protein concentration determined by the Brilliant Blue G dye-binding assay of Bradford using BSA as a standard. For the nuclear protein, the extraction was performed using a Nuclear Extract Kit (Active Motif, UK), following the manufacturer’s instructions. Protein extracts were heated at 95°C for 5 min in loading buffer and loaded onto 10% SDS-polyacrylamide gels together with a molecular weight marker. After routine electrophoresis and transfer, the PVDF membrane was blocked with 5% fat free milk in PBST (0.05% Tween 20) for 1 h and incubated with a specific primary antibody in 5% milk in PBST for 1 h. The membrane was washed three times for 45 min with PBST and then incubated with the secondary antibody diluted with 5% milk in PBST for 1 h. After further washing the membrane three times for 45 min with PBST, antibody binding was determined by a Chemiluminescence detection kit and densitometry was measured by Fluor Chem Imager (Alpha Innotech, San Leandro, CA).

To detect FANCD2, whole cell extracts prepared as described previously [47 (link), 48 (link)] were resolved in pre-cast Tris-acetate gels (Life Technologies), transferred onto nitrocellulose membranes and probed with the α-FANCD2 antibody ab2187 (Abcam). Mouse α-CHK1 antibody was from Santa Cruz (Cat. No. sc-8408). Phosphorylation of CHK1 and CHK2 was analyzed with a Phospho-Chk1/2 Antibody Sampler Kit (Cell Signaling, Cat. No. 9931). Other antibodies were: α-PCNA Cat. No. sc-56 (Santa Cruz), α-RPA32 Cat. No. A300-244A (Bethyl Laboratories), α-γH2AX Cat. No. 05-636 (Millipore), α-Nucleolin Cat. No. 396400 (Life technologies). Biotin-conjugated EdU was visualized in dot blots with HRP-conjugated α-biotin antibody, Cat. No. 7075 (Cell Signaling). All proteins were visualized by ECL (Amersham or Thermo Scientific) and quantified using Storm Phosphoimager and ImageQuant software (Molecular Dynamics) or FluorChem Imager (Alpha Innotech), using manufacturer-supplied software. For presentation, images were saved in TIFF format, adjusted for brightness/contrast and cropped using Adobe Photoshop, and assembled into figures in CorelDraw. Brightness/contrast adjustments were made to entire images. In some cases an image of one and the same blot was cut and spliced to delete extraneous lanes or to change the order of lanes.

The activation status of various cellular factors was assessed by utilizing a Protein-DNA Array I (Panomics) exactly as previously published [69 (link)]. Briefly, nuclear extract was isolated from 10⁶ cells and mixed with biotinylated probes to allow the formation of protein-DNA complexes. Labeled probes were eluted from free probes, hybridized to the pretreated array membranes and scanned using the FluorChem Imager (Alpha Innotech). Spots were quantified by Image J software and normalized to their respective controls after background subtraction. The key identified factors from arrays were verified by Western blotting for phosphorylated p38 MAPK (Life Technologies), phospho-ERK5, phospho-Smad2, phospho-Akt, and phophoMEF-2A (Cell Signaling).