Fanci

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.

The antibody against chicken Atm was raised by Pocono Rabbit Farm (Canada); ATM human antibody, SNF2H, BAZ1A, RIF1, and FANCI were all purchased from Bethyl Laboratories; and the RSF1 monoclonal antibody, phospho-ATM-S1981, and γ-H2AX were from Millipore and NBS1 antibody from Novus. Human ATR and FANCD2 antibody were from Santa Cruz. Histone H3.1, Actin, and BAZ1B were purchased by Abcam. CENPA antibody was a kind gift from Dr. Kevin Sullivan. MHF1 and MHF2 were a kind gift from Dr. Weidong Wang (Baltimore, Maryland), and the FANCD2 antibody was a kind gift from Professor Minoru Takata (Kyoto, Japan).

Cells were harvested following various treatments, lysed in lysis buffer with phosphatase inhibitors (Roche), and subjected to western blotting analysis and immunoprecipitation as described in ref. ⁴³ , using various antibodies as follows: FANCD2 (Santa Cruz, sc-20022), FANCI (Bethyl, A300-212A), FANCI (Proteintech, 20789-1-AP), FANCL polyclonal rabbit antibody (gift from Dr. Weidong Wang), FLAG (Sigma-Aldrich, F1804), β-actin (Sigma-Aldrich, A5441), p62 (MBL Life science, PM045), p21 (Cell Signaling Technology, 2947S), and Beclin 1 (Cell Signaling Technology, 4122S). RPS27L and RPS27 polyclonal rabbit antibodies were raised and purified as described in ref. ^{10 (link)}. Anti-FLAG M2 affinity gel (Sigma-Aldrich, A2220) were used to immunoprecipitate ectopically expressed FLAG-tagged proteins.