Protein a agarose beads

HEK 293T cells were lysed in lysis buffer (50 mm Tris–Cl (pH 7.4), 150 mm NaCl, 0.3% Igepal CA‐630, 0.2% Triton X‐100, 10 mm NaF, 1 mm sodium orthovanadate, and protease inhibitors). Cell lysates were incubated with 10 μL protein A‐agarose beads (Invitrogen, Carlsbad, CA, USA) for 1 h at 4 °C to remove nonspecific binding. Precleared lysates were incubated with anti‐FANCA antibody (A301‐980A; Bethyl Laboratories, Montgomery, TX, USA) for 18 h, followed by 10 μL protein A‐agarose beads for an additional 3 h at 4 °C. Beads were washed twice with lysis buffer and subjected to SDS/PAGE. The presence of Aurora A kinase was detected via immunoblotting with an anti‐Aurora A kinase antibody (Abcam, Cambridge, MA, USA).

HEK 293T cells were transfected with pcDNA3-V5-PRKAA1 and pcDNA3-HA-FANCG or pcDNA3-HA-FANCA or the pcDNA3 empty vector using the Effectene Transfection Reagent (Qiagen, Valencia, CA, USA). The cells were treated 1 day later with 200 ng/mL MMC for 16 h and then lysed in lysis buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 0.3% Igepal CA-630, 0.2% Triton X-100, 10 mM NaF, 1 mM sodium orthovanadate, and protease inhibitors). Co-immunoprecipitation was performed as described previously [17 (link)]. Briefly, cell lysates were precleared with 10 μL protein A-agarose beads (Invitrogen, Carlsbad, CA, USA) and incubated with anti-HA antibody-conjugated agarose beads (Sigma, St. Louis, MO, USA), anti-V5 antibody-conjugated agarose beads (Sigma), or an anti-FANCA antibody (A301-980A, Bethyl Laboratories, Montgomery, TX, USA) with protein A-agarose beads for 18 h at 4°C. The beads were washed with lysis buffer and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Co-immunoprecipitated proteins were detected by immunoblotting with anti-V5 (Invitrogen), anti-FANCA (Bethyl Laboratories), anti-FANCG (Novus Biologicals, Littleton, CO, USA), or anti-AMPKα (Cell Signaling Technology, Danvers, MA, USA) antibodies.