Np 40

Cells were cultured on 12 mm diameter microscope cover glasses, permeabilized with PBS containing 0.1% Triton X-100 for 3 min, and then fixed with 3.7% formaldehyde (Sigma) for 10 min at room temperature. Cells washed by PBS were extracted with 0.5% NP-40 (USBiological, Salem, MA, USA) for 5 min at room temperature. After they were washed, cells were blocked with blocking buffer (0.2% gelatin and 0.5% BSA in PBS) for 1~2 h at room temperature and then incubated with primary antibodies, such as FANCA (Merck Millipore: MABC557), FANCD2 (Novus Biologicals: NB100-182), PML (Santa Cruz Biotechnology: sc-966), or γH2AX (Cell Signaling Technology: #2595) overnight at 4 °C. Samples were washed three times for 10 min with blocking buffer. Secondary antibodies (Abcam, Cambridge, UK) mouse 488 (Abcam: ab150109), rabbit 594 (Invitrogen: A21207), mouse 594 (Abcam: ab150112), and rabbit 488 (Abcam: ab150061) were diluted 1:2500 and incubated for 1 h at room temperature in the dark. Finally, cells were washed three times for 30 min and mounted in Vectashield^® containing DAPI (Vector Laboratories, Burlingame, CA, USA).

At indicated time points post-transfection, the cells were centrifuged, washed with PBS, and lysed on ice for 30 min in lysis buffer (10 mM Tris (Thermo Fisher Scientific; Waltham, MA, USA), pH 8.0, 120 mM NaCl (Thermo Fisher Scientific), 0.5% NP-40 (US Biological; Salem, MA, USA), 1 mM EDTA (Thermo Fisher Scientific) with protease inhibitors (0.5 M phenyl methyl sulfonyl fluoride (PMSF), 1 mg/mL leupeptin, 1mg/mL pepstatin) (Sigma; St. Louis, MO, USA). Equal amounts of protein were loaded and electrophoresed on 10% SDS–polyacrylamide gel. The proteins were transferred onto PVDF membrane (Immobilon transfer membrane, Millipore; Burlington, MA, USA). After electroblotting, the membranes were blocked with Tris-buffered saline with Tween 20 (1 M Tris–HCl (Thermo Fisher Scientific), pH 7.5, 150 mM NaCl, and 0.5% Tween 20 (Thermo Fisher Scientific) containing 2% non-fat dry milk. Primary antibodies recognizing Cx43 or α-tubulin were diluted in blocking buffer and incubated for 30 min. The membranes were then washed, incubated with the appropriate secondary antibodies in a blocking buffer for 30 min, and washed again. The blotted proteins were detected using enhanced chemiluminescence detection system (0.1 M Tris, pH 8.5 (Thermo Fisher Scientific), 12.5 mM luminol (Sigma), 0.2 mM p-coumaric acid (Sigma), 10 μL 30% hydrogen peroxide (Thermo Fisher Scientific).