Irdye 680 conjugated anti mouse igg

Protein was extracted from the fresh ACP surgical specimens by lysing cells with protease inhibitor cocktail (Roche, USA). After measuring the protein content with a Bradford assay, 20 μg of protein was resolved by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% skim milk in TBST for 2 h at room temperature and incubated overnight with the following antibodies: anti-HAT, anti-HDAC1, anti-HDAC2, anti-HDAC3, anti-HDAC8 (1:1000, Abcam, USA), anti-CBX4, anti-Runx2, anti-histone (1:1000, Cell Signaling Technology, USA), and anti-β-actin (1:1000, Proteintech, USA). The specimens were then incubated with secondary antibodies, IRDye800-conjugated anti-rabbit IgG and IRDye680-conjugated anti-mouse IgG (1:15,000, LiCor, USA), for 1 h at room temperature to label the primary antibody. An Odyssey Infrared Image System (LiCor, USA) was used to analyze signal intensities. The densitometry results were first normalized to the density obtained for β-actin or histone and then compared with that of the control to obtain relative fold changes.

Total protein was extracted from exponentially growing cells at passage 8–10 and 40 g/mL were resolved by SDS-PAGE using a 4–15% gradient gel (Bio-Rad Laboratories). After transfer and blocking overnight at 4 °C in Odyssey Blocking Buffer (Li-Cor, Lincoln, NE, USA) proteins were detected by primary antibodies against BRCA1 [2A-9] (1:500, kindly provided by Stephen Smith, Leibnitz Institute, Jena, Germany), ATR [N-19] (Santa Cruz, St. Cruz, CA, USA, 1:1000), CHK1 [2G1D5] (Cell Signaling, Danvers, MA, USA, 1:750), RAD51 [14B4] (1:2.000, GeneTex, Irvine, CA, USA), MRE11A [12D7] (Abcam, Cambridge, UK, 1:500), pCHK1 [Ser296] (Cell Signaling, 1:1000), -actin [AC-74] (1:50.000, Sigma, St. Louis, MO, USA). Primary antibodies were detected with IRDYE 680 conjugated anti-mouse IgG, IRDYE 800 conjugated anti-rabbit IgG (Li-Cor, 1:7500), IRDYE 680 conjugated anti-rabbit IgG (Licor, 1:7.500 or 15.000) or IRDYE 800 conjugated anti-mouse IgG (Li-Cor 1:7.500 or 15.000). Quantitative and qualitative analysis was done by using Li-Cor Odyssey (Li-Cor, Lincoln, NE, USA).