Fanca

Primary antibodies used were FANCA (A301-980A) (1:2000), FANCD1 (A300-005A) (1:1000), FANCD2 (A302-174A) (1:2000), FANCE (A302-125A) (1:1000), FANCI (A300-212A) (1:700), FANCM (A302-637A) (1:1000), FANCO (A302-645A) (1:1000), FANCP (A302-270A) (1:1000), FANCQ (A301-315A) (1:1000), H2AX (A300-082A) (1:5000) from Bethyl Laboratories; FANCF (PA5-18202) (1:1000), FANCG (PA5-27117) (1:1000), FANCL (PA5-19332) (1:1000), PIK3C2A (MA526505) (1:1000), and TFRC (136800) (1:1000) were from ThermoFisher; β-actin (sc-47778) (1:2000) was from Santa Cruz Biotechnology; FANCS (OP92-100UG) was from Calbiochem; γ-H2AX (NB100-78356) (1:5000) was from Novus Biologicals. Secondary antibodies used were goat anti-rabbit IgG-HRP (sc-2357) (1:5000), goat anti-mouse IgG-HRP (sc-2005) (1:5000), and goat anti-rat IgG-HRP (sc-2303) (1:5000) were from Santa Cruz Biotechnology.

Cells were harvested and resuspended in protein extraction buffer (50 mM Na₃PO₄, 300 mM NaCl, 0.5% NP-40, 1 mM DTT, 1× PI cocktail). Cells were then sonicated, centrifuged at 4 °C, and supernatants were analyzed for protein concentration using the Bradford method. 20–80 μg protein were loaded in each well of a polyacrylamide gel after denaturing 5 min at 95 °C. Gels were run 1–1.5 h at 150 V then transferred to nitrocellulose membranes, blocked for 1 h with 5% milk, then probed with the following antibodies: FANCA (Bethyl-1:1000), FAAP20 (Sigma, Weidong Wang-1:250), FANCG (Santa Cruz-1:50), Actin (Santa Cruz-1:2000), H2B (Cell Signaling Technologies-1:1000), BRCA2 (Sigma, OHSU-1:500), RAD51 (BioAcademia-1:500), HSP90 (Santa Cruz-1:2000), FANCD2 (Proteintech-1:1000). Membranes were then washed 2× with TBST, probed with secondary antibody for 1 h (anti-Mouse HRP, anti-Rabbit HRP, Sigma), washed 3× with TBST, then developed using peroxide treatment and autoradiography.