Goat anti actin

The determination NO₂ in cells supernatants was carried out using the quantitative Nitric Oxide Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), following the instructions by the manufacturer_. The determination of iNOS was made as follows: Cells were lysed on ice in lysis buffer (1% NP-40, 50 mN Tris-HCl, pH 7.6, 150 mM NaCl and 2 mM EDTA) and clarified by centrifugation at 5000× g for 5 min in a microcentrifuge at 4 °C. In addition, LPS infected macrophages were used as the positive control for this experiment. Proteins were separated by SDS-PAGE and transferred to polyvinylidine fluoride (PVDF) membranes (Biorad, Hercules, CA, USA). Antibodies used were rabbit anti-iNOS (Cell Signaling, Danvers, MA, USA) and goat anti-actin (Abcam, Cambridge, UK). Chemiluminescence was detected using the enhanced chemiluminescent (ECL) reagents (GE Healthcare, Chicago, IL, USA). To determine iNOS expression, densitometry was performed using Adobe Photoshop.

The following antibodies were used: mouse anti-MiTF (Abcam), mouse anti-FANCD2 (Santa Cruz), rabbit anti-FANCD2 (Abcam), rabbit anti-FANCA (Bethyl), mouse anti-Flag (Sigma), rabbit anti-p53 serine 15 (Cell Signaling Technology), mouse anti-p53 (Santa Cruz), rabbit anti p27 (Cell Signaling), rabbit anti-p21 (Santa Cruz), mouse anti-Cyclin-A (Abcam), rabbit anti-53BP1 (Abcam), mouse anti-gH2AX (Millipore), mouse anti-alpha-tubulin (Abcam), mouse anti-vinculin (Abcam), and goat anti-actin (Abcam).