iPOND Assay with PAA Treatment

1×10⁸ HH514-16 cells were induced with 3mM NaB with or without phosphonoacetic acid (PAA) (200 μg/ml) for 36 hours prior to performing iPOND as described previously [29 (link)]. Briefly, cells were pulsed with 10 μM EdU for 15 min, spun down, cross-linked with 1% formaldehyde for 20 min, and quenched with 0.125 M glycine for 5 min. For click chemistry, cells were incubated with 10 μM biotin-azide in click reaction buffer for 2 hours. Nuclei were isolated and digested with 1 μL of micrococcal nuclease (10011, Cell Signaling) at 37°C for 20 min and suspended in cold lysis buffer (1% SDS, 50 mM Tris, pH 8.0), and subjected to sonication using a microtip sonicator to break nuclear membranes. After removing debris, the supernatant was incubated with 100 μL of streptavidin agarose beads (69203, EMD Millipore) overnight at 4°C and washed three times with lysis buffer and one time with 1M NaCl. Protein-DNA complexes were eluted with 2X Laemmli buffer at 95°C for 25 min and subjected to immunoblotting.